Effects of prostaglandin E2, indomethacin and adenosine deaminase on basal and insulin-stimulated glucose metabolism in human adipocytes

The effects of prostaglandin E2 were studied on glucose metabolism (3-O-methylglucose transport, CO2 production and lipogenesis) in human adipocytes. Initially, the effects of endogenously produced adenosine and prostaglandins were indirectly demonstrated by using adenosine deaminase and indomethacin in the incubations. From these studies it was found that adenosine deaminase (5 micrograms/ml) had a pronounced effect on adipocyte glucose metabolism in vitro. In the basal (nonhormonal-stimulated) state, glucose transport, CO2 production and lipogenesis were inhibited by about 30% (P less than 0.05). Furthermore, adenosine deaminase significantly inhibited the isoproterenol- and insulin-stimulated CO2 production and lipogenesis (P less than 0.01). Indomethacin (50 microM) had a consistently inhibitory effect on the insulin-stimulated CO2 production (P less than 0.05), whereas indomethacin had no significant effects on basal or isoproterenol-stimulated glucose metabolism. In contrast to the relatively minor effect of endogenous prostaglandins, the addition of exogenous prostaglandin E2 significantly stimulated the glucose transport, glucose oxidation and lipogenesis in human adipocytes, especially in the presence of adenosine deaminase. Half-maximal stimulation was obtained at prostaglandin E2 concentrations of 2.2, 0.8 and 0.8 nM, respectively. The effect of prostaglandin E2 was specific, since the structurally related prostaglandin, prostaglandin F2 alpha, had practically no effect on glucose metabolism. The maximal effect of prostaglandin E2 (1 microM) on glucose metabolism was 30-35% of the maximal insulin (1 nM) effect. When insulin and prostaglandin E2 were added together, the effect of prostaglandin E2 on glucose metabolism was additive at all insulin concentrations tested.     

Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.     

Interleukin-1 beta is a potent stimulator of prostaglandin synthesis in bovine luteal cells.

Interleukin-1 (IL-1) is a polypeptide that has both local and systemic effects on numerous tissues, including endocrine cells. To evaluate the effect of IL-1 on luteal function, bovine luteal cells were cultured for 5 days with increasing concentrations (0.1, 0.5, 1.0, 2.5, 5.0, 10.0 ng/ml) of recombinant bovine interleukin-1 beta (rbIL-1 beta). IL-1 beta increased the production of luteal 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2), and prostaglandin F2 alpha (PGF2 alpha) in a dose-dependent manner, but had no effect on progesterone (P4) production. Treatment with the cyclooxygenase inhibitor, indomethacin (5 micrograms/ml), inhibited basal, as well as rbIL-1 beta-stimulated prostaglandin production. Addition of Iloprost (a synthetic analogue of prostacyclin, 5 ng/ml) suppressed basal production of PGF2 alpha and PGE2, but did not reduce the stimulatory effect of rbIL-1 beta. Similarly, PGF2 alpha suppressed basal, but not IL-1 beta-stimulated, production of 6-keto-PGF1 alpha. PGE2 had no effect on the synthesis of either PGF2 alpha or 6-keto-PGF1 alpha. P4 (1.75 micrograms/ml) reduced basal as well as rbIL-1 beta-stimulated production of 6-keto-PGF1 alpha, PGE2, and PGF2 alpha. These results indicate that IL-1 beta could serve as an endogenous regulator of luteal prostaglandin production. It appears that IL-1 beta action is not modified by exogenous prostaglandins, but is at least partially regulated by elevated P4. It is possible that the role of IL-1 beta in stimulation of luteal prostaglandin production may be confined to a period characterized by low P4 levels, such as during luteal development or regression.     

Plasma thromboxane B2 levels and thromboxane B2 production by platelets are increased in patients during spinal and epidural anesthesia

Concentrations of thromboxane (Tx) B2 in plasma and its production by platelets were measured in 20 spinal and 10 epidural anesthesia patients scheduled for small operations in the lower extremities. The main metabolite of prostacyclin, 6-keto-PGF1 alpha and prostaglandin (PG) E2 in plasma were also determined. Plasma TxB2 and TxB2 production by platelets increased during both spinal and epidural anesthesia. Plasma TxB2 levels also remained elevated 1 h after anesthesia. The plasma concentrations of 6-keto-PGF1 alpha and PGE2 did not change during spinal or epidural anesthesia. In in vitro studies, only low concentrations of lidocaine (0.5-1.0 micrograms/ml) and bupivacaine (0.5-3.0 micrograms/ml) increased platelet TxB2 production. In platelet rich plasma, neither lidocaine nor bupivacaine in concentrations of 0.5-3.0 micrograms/ml caused constant changes in ADP-induced platelet aggregation, but they inhibited it in toxic concentrations (12 micrograms/ml). The results suggest that the increased TxB2 plasma levels and platelet TxB2 production during regional anesthesia are not caused by local anesthetics itself but by other factors, e.g. tissue trauma. In clinically found concentrations, local anesthetics do not cause any constant changes in platelet aggregation.     

Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.     

Differential inhibition of human placental prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestation and found that this antagonist (1 microgram/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 micrograms/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

In vitro inhibition of prostaglandin production by azathioprine and 6-mercaptopurine

Although there are many data concerning the cytotoxic and immunosuppressive effects of antimetabolites such as azathioprine and 6-mercaptopurine, the mechanism of their antiinflammatory action has not been extensively investigated. In the present work, it is shown that azathioprine and 6-mercaptopurine (10-500 micrograms/ml) inhibit in a dose-dependent manner the production of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 by unseparated spleen cells as well as that of 6-keto-PGF1 alpha by adherent peritoneal macrophages. This inhibitory effect appears rapidly in vitro (within 15 min of incubation) and is observed in the presence of exogenous arachidonic acid (5 x 10(-6) M). The persistence of this effect in the presence of arachidonic acid, together with the fact that the production of four cyclooxygenase derivatives of acid arachidonic metabolism are inhibited, suggests that these drugs are acting at the cyclooxygenase level. The finding that cytotoxic and immunosuppressive agents, which act mainly by inhibiting RNA and DNA synthesis, can block prostaglandin production, may explain part of their antiinflammatory effects.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

Uterine vein prostaglandin levels in late pregnant dogs.

We studied the uterine venous plasma concentrations of prostaglandins E2, F2 alpha, 15 keto 13,14 dihydro E2 and 15 keto 13,14 dihydro F2 alpha in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E2 and F2 alpha in pregnancy in vivo. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E2 and 15 keto 13,14 dihydro E2 were 1.35 +/- .27 ng/ml and 1.89 +/- .37 ng/ml, respectively; however, we could not find any prostaglandin F2 alpha and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F2 alpha and E2 from endoperoxides, prostaglandin F2 alpha production in vivo must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E2 is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F2 alpha does not appear to play a role at this stage of pregnancy.     

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.     

Single-turnover and steady-state kinetics of hydrolysis of cephalosporins by beta-lactamase I from Bacillus cereus.

Purified mast cells derived from rat peritoneal fluids and dog mastocytomas were extracted with 1 M-NaCl and sonication techniques. The mast-cell products increased the production of mononuclear cell factor from human peripheral blood mononuclear cells in culture, as judged by the enhanced stimulation of prostaglandin E (2-5 fold) and collagenase (3-11-fold) production by cultured adherent synovial cells. Heparin alone (1-10 micrograms/ml) induced a similar stimulation of mononuclear-cell-factor production by monocyte cultures, whereas histamine (1-10 micrograms/ml) had no effect. The stimulatory effect of mast-cell products and heparin represented a direct effect on mononuclear cells; they did not potentiate the effect of monokine on the synovial cells. These results suggest that mast-cell-macrophage interactions may play a significant role in the pathogenesis of inflammation and connective-tissue degradation.     

Stimulatory effect of prostaglandin E2 on 1 alpha,25-dihydroxyvitamin D3 synthesis in rats.

The effect of prostaglandin E2 on accumulation in plasma of 1 alpha,25-dihydroxy[3H]vitamin D3 from 25-hydroxy[3H]vitamin D3 was studied in vivo using vitamin D-deficient thyroparathyroidectomized rats. Intra-arterial infusion of 10-50 micrograms of prostaglandin E2/h caused a significant stimulation of 1 alpha,25-dihydroxy[3H]vitamin D3 production. No significant changes in plasma Ca2+ and Pi concentrations or urinary cyclic AMP excretion were observed after prostaglandin E2 infusion. Theophylline did not enhance the effect of a submaximal dose of prostaglandin E2 on 1 alpha,25-dihydroxy[3H]vitamin D3 production. These data indicate that prostaglandin E2 stimulates plasma accumulation of 1 alpha,25-dihydroxy[3H]vitamin D3 independent of the adenylate cyclase/cyclic AMP system, and suggest that prostaglandin E2 has a modulatory role in the regulation of 25-hydroxyvitamin D3 1 alpha-hydroxylase in the kidney.     

Effects of prostaglandin E2 and F2 alpha on peri-implantation development of mouse embryos in vitro.

The effects of exogenous prostaglandin (PG) E2 and F2 alpha on the morphology and lactate dehydrogenase (LDH) activities of pre-implantation mouse embryos in vitro were studied. A 24-hour exposure from 0.01 to 10 micrograms/ml of PGE2 at the 4-cell or morula stages had no effect on the morphology of embryos during the 144 hours in culture. Exposure to 10 micrograms/ml PGE2 at the blastocyst stage accelerated and enhanced spreading of the trophoblast in vitro. Embryos treated at 0.01 to 10 micrograms/ml PGE2 at various stages all showed a more rapid decline in LDH activity from morula to blastocysts. Treatment with 50 or 100 micrograms/ml PGE2 led to abnormal morphology of embryos in vitro. In contrast, continuous treatment with 0.01 to 100 micrograms/ml PGF2 alpha from 4-cell to early post-implantation (day 8) had no effect on the morphology of embryos, although breakdown of LDH was again accelerated. These results suggest that the peak of PGE2 secretion on day 4 of pregnancy in mice may enhance trophoblastic outgrowth, and the lower levels of PGE2 and PGF2 alpha secreted earlier in pregnancy modulate the development of pre-implantation mouse embryos.     

Potentiation of the natriuretic effect of clonidine following indomethacin in the rat.

Previous studies have demonstrated a diuretic effect of clonidine at low intrarenal infusion rates with a natriuretic effect being observed at high infusion rates (greater than or equal to 3 micrograms.kg-1.min-1). The natriuresis at high infusion rates may have been secondary to increased renal prostaglandin production. We therefore evaluated the effects of indomethacin (a cyclooxygenase inhibitor) on the response to clonidine in the anesthetized rat. Intrarenal infusions of saline (vehicle) or clonidine (0.1, 0.3, 1, and 3 micrograms.kg-1.min-1) were examined both in the presence and absence of pretreatment with indomethacin (5 mg/kg, i.p.). Clonidine produced a dose-related increase in urine volume and free water clearance at 0.3, 1, and 3 micrograms.kg-1.min-1 as compared with the vehicle group. Sodium excretion and osmolar excretion were increased only at the highest infusion rate investigated. Following indomethacin pretreatment, clonidine produced a greater increase in urine volume at each infusion rate investigated. The indomethacin pretreatment also resulted in a potentiation of the natriuretic effect of clonidine at all infusion rates. Interestingly, this was associated with an increase in osmolar clearance but not free water clearance. These effects of indomethacin were reversed by infusion of prostaglandin E2. An infusion of prostaglandin E2 attenuated the indomethacin-induced increase in both urine flow rate and sodium excretion, indicating that the effects of indomethacin were mediated by prostaglandin inhibition. These results suggest that endogenous prostaglandin production attenuates the renal effects of clonidine, and as well, that in the presence of alpha 2-adrenoceptor stimulation, prostaglandin E2 mediates an antidiuretic and antinatriuretic effect.     

Inhibition of interleukin 2 production by prostaglandin E2 is not absolute but depends on the strength of the stimulating signal

In view of the eminently important role of interleukin-2 (IL-2) in T-cell responses, and in view of reports about immune stimulatory effects of PGE2, we reinvestigated the question whether PGE2 inhibits IL-2 production. It was found that PGE2 does not inhibit IL-2 production in murine spleen cell cultures after optimal stimulation (5 micrograms/ml concanavalin A) but does inhibit at suboptimal stimulation conditions. The failure of PGE2 to inhibit IL-2 production at optimal concanavalin A concentration was demonstrated by two independent IL-2 assays namely by the co-stimulator assay and by the proliferation of IL-2-dependent T-cell clone W-2. Our observations indicated that the inhibitory effect of PGE2 depends on the strength of the stimulating signal. IL-2 production in cultures with 5 micrograms/ml concanavalin A was also not suppressed by PGE1, by prostaglandin D2, thromboxane B2 (T X B2), and prostaglandin F2.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.     

Anisodamine inhibits shiga toxin type 2-mediated tumor necrosis factor-alpha production in vitro and in vivo

Cytokines, in particular tumor necrosis factor (TNF), appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection. In this study we examined the effect of anisodamine, a vasoactive drug, on TNF-alpha production in Shiga toxin type 2 (Stx2)-stimulated human monocytic cells in vitro and in Stx2-injected mice sera in vivo. Human monocytes and THP-1 cells were stimulated by Stx2 (1-100 ng/ml) with or without anisodamine addition (1-400 micrograms/ml). For in vivo evaluations, C57BL/6 mice were given a single intraperitoneal injection of anisodamine (6-50 mg/kg) or saline after intraperitoneal injection of Stx2 (50 ng/kg). The results showed that anisodamine suppressed Stx2-induced TNF-alpha production in a dose- and time-dependent manner. Anisodamine also suppressed Stx2-induced TNF-alpha mRNA expression. Further study showed that endogenous prostaglandin E2 may be involved in this inhibitory effect. In contrast to TNF-alpha mRNA, anisodamine at concentrations as high as 400 micrograms/ml did not decrease Stx2-induced IL-1 beta and IL-8 mRNA levels. In addition, anisodamine (> 50 micrograms/ml) increased Stx2-stimulated THP-1 cell viability. Levels of TNF-alpha in anisodamine-treated mice sera were significantly lower than those in the saline-treated group 1.5 and 24 hr after Stx2 injection. Anisodamine induced a lower percentage of death in Stx2-injected mice. Taken together, our results indicate that anisodamine has an important regulatory effect on Stx2-induced TNF-alpha production in vitro and in vivo. The present study suggested that this drug should be further investigated for its effects on Stx2-mediated diseases in humans.     

Evidence for suppression of serum LH without elevation in serum estradiol or prolactin with a brain-enhanced redox delivery system for estradiol

We developed a redox system for brain-enhanced delivery of estradiol based on an interconvertible dihydropyridine in equilibrium pyridinium salt carrier. Estradiol (E2), when combined with the lipoidal carrier, readily crosses the blood-brain barrier. The carrier, when oxidized, reduces the rate of exit of the estradiol-carrier complex from the brain. Subsequent hydrolysis of the carrier provides sustained production of estradiol in the brain. The aim of the study was to evaluate the effects of single vs. multiple injections of the estradiol-chemical delivery system (E2-CDS) on both central and peripheral estrogen-responsive tissues. Ovariectomized Sprague-Dawley rats received an intravenous injection of E2-CDS at 10, 33, 100 or 333 micrograms/kg BW or the drug vehicle, dimethyl sulfoxide (DMSO; 0.5 ml/kg) every 2 days for 7 injections (2 weeks) or a single injection only at 2 days before sacrifice. With a single injection, E2-CDS did not affect serum luteinizing hormone (LH) levels at the 10 micrograms/kg dose but caused a dose-dependent reduction in serum LH of 39-52% at the dose range of 33 to 333 micrograms/kg. By contrast, multiple injections of E2-CDS caused a 32 to 76% reduction in serum LH levels at doses ranging from 10 micrograms/kg to 333 micrograms/kg. Additionally, multiple doses of E2-CDs caused a dose-dependent reduction in body weight at the 10 and 33 micrograms/kg doses with the higher doses causing no further weight reduction. For both single and multiple dosage groups, serum E2 levels remained unchanged after doses of E2-CDS of 10 and 33 micrograms/kg, then increased to 21 pg/ml for the single dosage group and to 23 pg/ml for the multiple dosage group at the 100 micrograms/kg dose, and to 59 pg/ml for singly-injected rats and 60 pg/ml for multiply-injected rats at the 333 micrograms/kg dose. Serum prolactin concentrations were closely correlated with serum E2 levels for both the single and multiple dose groups. These data reveal that a single or multiple doses of E2-CDS can reduce serum LH levels without elevating serum E2 or prolactin concentrations, supporting the concept of brain-enhanced delivery of estradiol with an estradiol chemical delivery system.     

Novel effects of PGF2 alpha on airway function in asthmatic subjects

The effects of inhaled prostaglandin F2 alpha (PGF2 alpha) have been examined in eight subjects with asthma. Incremental PGF2 alpha aerosol concentrations, ranging from 1 to 5,000 micrograms/ml, were administered at 15-min intervals. Plethysmographic specific airway conductance (sGaw), forced expiratory volume at 1 s (FEV1), and maximum expiratory flow at 50% vital capacity breathing air (Vmax50% air) and 80% He-20% O2 (Vmax50% He-O2) were measured after each dose and compared with saline control values. We observed unexpected triphasic dose-response characteristics, i.e., an initial decline in physiological variables at low concentrations (1-100 micrograms/ml), followed by improvement at intermediate concentrations (100-1,000 micrograms/ml) and a subsequent steep decline at high concentrations (1,000-5,000 micrograms/ml). Improvement in FEV1 and Vmax50% air between 100 and 1,000 micrograms/ml was associated with sGaw increases above control levels in six subjects and a significant fall in density-dependent index (Vmax50% He-O2/Vmax50% air) when compared with values before challenge and at low concentrations. Inhaled atropine (5 mg) improved prechallenge lung function but had no effect on PGF2 alpha dose-response characteristics. Intermediate PGF2 alpha concentrations given as a single dose consistently induced greater FEV1 reductions than the same concentration during graded dose challenges. Our findings are consistent with the demonstration of in vivo airway tachyphylaxis and indicate that airway effects of PGF2 alpha are far more complex than previously reported. Moreover, these novel effects suggest that, in addition to its well-known bronchoconstrictor effects, PGF2 alpha directly or indirectly causes airway relaxation, predominantly in large airways.