Age and sex effects on the pharmacokinetics of linezolid.

OBJECTIVE: To evaluate the possible effects of age and sex on the pharmacokinetics of linezolid in healthy volunteers. METHODS: A single 600-mg dose of linezolid was administered orally to young (18-40 years) and elderly (> or =65 years) healthy males and females. Blood and urine samples were collected until 48 h after dosing and assayed for linezolid concentrations using a validated high-performance liquid chromatography method. Pharmacokinetic parameters were assessed using noncompartmental methods. Comparisons of the pharmacokinetic parameters for each age and sex group were performed using a two-way analysis of variance model. Pairwise comparisons were done using least-square means analysis. RESULTS: Peak plasma drug concentrations occurred within 1.5 h after linezolid administration for males and females in both age groups. However, the maximum concentration achieved differed significantly between males and females. There was no significant difference between males and females or young and elderly for mean apparent elimination rate constant or half-life. There was no difference in mean apparent oral clearance (CLPO) between the young and elderly; however, there was a significant difference between males and females. Mean CLPO for females was approximately 37% less than mean CLPO for males when not corrected for body weight. Correcting for differences in weight reduced this difference to approximately 20%. Overall, females had a slightly lower volume of distribution than males, but this was not affected by the age of subjects. CONCLUSIONS: A dose adjustment based on age and sex is not warranted due to the wide range of linezolid concentrations that are well tolerated and the relative small difference in linezolid disposition between males and females.

Linezolid absolute bioavailability and the effect of food on oral bioavailability.

Linezolid is a novel oxazolidinone antibiotic that has a spectrum of activity encompassing a variety of Gram-positive bacteria. The objectives of this study were twofold: (1) to compare the absorption of linezolid tablets given immediately following a high-fat meal with the absorption of tablets administered while fasting, and (2) to assess the bioavailability of a 375-mg oral dose given while fasting relative to a 375-mg dose of linezolid sterile solution given intravenously. Venous blood samples were taken over the 48 h following the single dose administration of both the oral and intravenous (IV) treatment. Samples were subsequently frozen for the determination of linezolid concentrations by HPLC. The only statistically significant difference between the fasted and the fed treatment was in peak plasma concentration, with the mean C(max) for fasted subjects being 23% greater than that for subjects after consumption of a high-fat meal. Comparable AUC(0-infinity) values were measured under both conditions, indicating that the overall extent of absorption is the same. Therefore, the difference in C(max), while statistically significant, should not affect the therapeutic efficacy of linezolid when it is administered with food. There were no statistically significant differences in AUC(0-infinity), CL or half-life between the fasted oral treatment and the intravenous treatment. As expected, C(max) was statistically different between the two treatments. However, the mean absolute bioavailability (F) of the tablet, using the IV sterile solution as the reference treatment, was 103% (+/-20%).

Linezolid, a novel oxazolidinone antibiotic: assessment of monoamine oxidase inhibition using pressor response to oral tyramine.

The primary objective of this study was to compare the effects of oral linezolid with moclobemide and placebo on the pressor response to oral tyramine. Secondary objectives were to determine possible mechanisms of the effect based on changes in the pharmacokinetics of tyramine and to evaluate alternative methods for quantifying the pressor effect. Subjects received linezolid (625 mg bid orally), moclobemide (150 mg tid orally), or placebo for up to 7 days. Using the oral tyramine dose producing a >30 mmHg increase in systolic blood pressure (SBP) (PD>30), a positive pressor response was defined as a PD>30 index (pretreatment/treatment ratio of PD>30) of > or = 2. There were 8/10, 11/11, and 1/10 responders with linezolid, moclobemide, and placebo, respectively. Responses returned to baseline within 2 days of drug discontinuation. The ratio of mean greatest SBP and heart rate at the time of greatest SBP (GSBP/HR) increased linearly with tyramine dose both pretreatment and during treatment with linezolid and moclobemide. During treatment, responses to tyramine when subjects took linezolid or moclobemide were significantly different from placebo. Both drugs significantly decreased tyramine oral clearance compared with placebo. Urinary excretion of catecholamines and metabolites was consistent with MAOI activity of the drugs, but results were variable. The MAOI activity of linezolid is similar to that of moclobemide, a drug used clinically without food restrictions. Restrictions to normal dietary intake of tyramine-containing foods are not warranted when taking linezolid.

Linezolid: pharmacokinetic and pharmacodynamic evaluation of coadministration with pseudoephedrine HCl, phenylpropanolamine HCl, and dextromethorpan HBr.

Linezolid is a novel oxazolidinone antibiotic with mild reversible monoamine oxidase inhibitor (MAOI) activity. The potential for interaction with over-the-counter (OTC) medications requires quantification. The authors present data evaluating the pharmacokinetic and pharmacodynamic responses to coadministration of oral linezolid with sympathomimetics (pseudoephedrine and phenylpropanolamine) and a serotonin reuptake inhibitor (dextromethorphan). Following coadministration with linezolid, minimal but statistically significant increases were observed in pseudoephedrine and phenylpropanolamine plasma concentrations; a minimal but statistically significant decrease was observed in dextrorphan (the primary metabolite of dextromethorphan) plasma concentrations. Increased blood pressure (BP) was observed following the coadministration of linezolid with either pseudoephedrine or phenylpropanolamine; no significant effects were observed with dextromethorphan. None of these coadministered drugs had a significant effect on linezolid pharmacokinetics. Minimal numbers of adverse events were reported. Potentiation of sympathomimetic activity by linezolid was judged not to be clinically significant, but patients sensitive to the effects of increased BP due to predisposing factors should be treated cautiously. No restrictions are indicated for the coadministration of dextromethorphan and linezolid.

A pharmacokinetic evaluation of concomitant administration of linezolid and aztreonam.

Linezolid, a new oxazolidinone antimicrobial agent, has a spectrum of activity encompassing a wide variety of Grampositive bacteria. The purpose of this study was to evaluate the pharmacokinetics of linezolid and aztreonam, an antimicrobial agent with selective activity against Gram-negative bacteria, when given alone and in combination. Healthy subjects were randomized to receive single, 30-minute intravenous infusions of (1) linezolid 375 mg, (2) aztreonam 1000 mg, and (3) linezolid 375 mg plus aztreonam 1000 mg in an open-label, crossover manner. The only statistically significant differences observed with combination treatment relative to each drug alone were an increase in the maximum plasma concentration of linezolid (approximately 18%) and an approximate 7% decrease in the apparent elimination rate of aztreonam, neither of which are expected to be clinically significant. In healthy subjects, the combination of linezolid and aztreonam was safe and well tolerated compared with each agent used alone. Pharmacokinetic data demonstrate that coadministration of linezolid and aztreonam does not alter the disposition of either agent under single-dose conditions. Therefore, it is not expected that a dose alteration of either agent will be necessary in a clinical setting.

Systemic absorption of clindamycin after intravaginal administration of clindamycin phosphate ovule or cream.

The absolute bioavailability of clindamycin phosphate vaginal ovule with comparison to a reference treatment of clindamycin phosphate sterile solution, as well as the relative bioavailability of the ovule compared to clindamycin phosphate vaginal cream, was evaluated in 12 healthy adult female volunteers. Subjects were randomly assigned to receive either the ovule or cream formulation intravaginally for 3 consecutive days during the two-way crossover portion of the study. During a third treatment period, all subjects received 100 mg of clindamycin as a 4-minute intravenous infusion of clindamycin phosphate sterile solution (10 mg/mL). Clindamycin concentrations in serum were assayed by a high-performance liquid chromatography method with detection by mass spectrometry. Pharmacokinetic analyses of the serum data indicated low systemic absorption of clindamycin from the vaginal cream (about 4%), consistent with results of previous bioavailability studies. Following intravaginal administration of the clindamycin phosphate ovule, systemic absorption averaged 30%, which was approximately sevenfold greater than after dosing with the vaginal cream. The higher drug absorption for the ovule may be related to differences in formulation effects on the vaginal membrane. Nevertheless, systemic exposure to clindamycin from the ovule is still considerably lower than from a therapeutic oral dose.

Hormonal effects on tirilazad clearance in women: assessment of the role of CYP3A.

This study assessed whether the previously reported difference in tirilazad clearance between pre- and postmenopausal women is reversed by hormone replacement and whether this observation can be explained by differences in CYP3A4 activity. Ten healthy women from each group were enrolled: premenopausal (ages 18-35), postmenopausal (ages 50-70), postmenopausal receiving estrogen, and postmenopausal women receiving estrogen and progestin. Volunteers received 0.0145 mg/kg midazolam and 3.0 mg/kg tirilazad mesylate intravenously on separate days. Plasma tirilazad and midazolam were measured by HPLC/dual mass spectrophotometry (MS/MS) assays. Tirilazad clearance was significantly higher in premenopausal women (0.51 +/- 0.09 L/hr/kg) than in postmenopausal groups (0.34 +/- 0.07, 0.32 +/- 0.06, and 0.36 +/- 0.08 L/hr/kg, respectively) (p = 0.0001). Midazolam clearance (0.64 +/- 0.12 L/hr/kg) was significantly higher in premenopausal women compared to postmenopausal groups (0.47 +/- 0.11, 0.49 +/- 0.11, and 0.53 +/- 0.19 L/hr/kg, respectively) (p = 0.037). Tirilazad clearance was weakly correlated with midazolam clearance (r2 = 0.129, p = 0.02). Tirilazad clearance is faster in premenopausal women than in postmenopausal women, but the effect of menopause on clearance is not reversed by hormone replacement. Tirilazad clearance in these women is weakly related to midazolam clearance, a marker of CYP3A activity.

Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): effects of body weight and injection sites on pharmacokinetics.

A new contraceptive option, medroxyprogesterone acetate (MPA) and estradiol cypionate (E2C) (MPA/E2C, Lunelle Monthly Contraceptive Injection), will soon be available for women in the US. This article reports the results of a US trial that assessed the effects of body weight and injection site on the pharmacokinetics of MPA, the progestin mediating contraceptive efficacy. This assessment was part of a nonrandomized, open-label, multicenter US study in healthy women receiving a monthly injection of MPA/E2C for 60 weeks. A total of 77 women (aged 18-47 years) at four centers participated in the pharmacokinetics assessment during the sixth or the seventh injection. For determination of serum MPA concentration-time profiles, blood samples were collected before the sixth and seventh injections (day 0) and on days 3, 7, 14, 21, and 28 after the sixth and seventh monthly administrations. For effects of injection site, MPA pharmacokinetics were compared at injection sites of the arm, hip, and leg. The pharmacokinetics of MPA, determined at the sixth and seventh injection, were not significantly affected by injection sites. The mean area under the curve (AUC0-28), however, was different between the arm and the leg injection sites; the difference was < 20%. More important, the average MPA trough concentrations (Cmin) at the fifth and sixth monthly injections were similar (range 0.42-0.51 ng/mL) for the three injection sites and well above the threshold levels of 0.10-0.20 ng/mL required to suppress ovulation. For effects of body mass index (BMI) on pharmacokinetics, women were stratified into three groups: thin/normal (BMI 18-28, n = 48), obese (BMI 29-38, n = 23), and highly obese (BMI > 38, n = 6). There were no significant differences in the pharmacokinetics of MPA among the three BMI categories. The only significant difference (p = 0.0387) was the AUC0-28 between BMI 18-28 and BMI 29-38. Because of the small sample size in the highly obese group, a reanalysis was performed by pooling subjects of the obese and highly obese groups. Results of the pooled statistical analysis remained the same. In summary, these results suggest that minor differences observed in the MPA pharmacokinetics--whether due to injection site or body weight or both--have no impact on the contraceptive efficacy of MPA/E2C, as trough concentrations (Cmin) are well above the threshold levels required to suppress ovulation. No dose adjustment is necessary based on body weight or injection site.

PIP: A nonrandomized, open-label, multicenter study assessed the effects of body weight and injection site on the pharmacokinetics of medroxyprogesterone acetate (MPA) and its progestin medicating contraceptive efficacy among 77 healthy, fertile women aged 18-47 years. For determination of serum MPA concentration-time profiles, blood samples were collected before the 6th and 7th injections (day 0) and on days 3, 7, 14, 21, and 28 after the 6th and 7th monthly administrations. For injection site effects, MPA pharmacokinetics were compared at the injection sites of the arm, hip, and leg; there was no significant finding. For effects of body mass, no significant differences were noticed in the pharmacokinetics of MPA among the 3 body mass indices (thin/normal, obese, highly obese). However, a difference (p = 0.0387) was found between normal and obese women (20%). The findings suggest that minor differences observed in MPA pharmacokinetics have no impact on the contraceptive efficacy of MPA/E2C, may it be due to injection sites or body weight, since trough concentrations are well above the threshold levels required to suppress ovulation.

Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): steady-state pharmacokinetics of MPA and E2 in surgically sterile women.

The steady-state pharmacokinetics and pharmacodynamics of medroxyprogesterone acetate (MPA) and estradiol (E2, released from E2C by esterase enzymes) were characterized after administration to surgically sterile women. This report describes the pharmacokinetics of this multiple-dose and open-labeled study (pharmacodynamics are reported in a subsequent article in this issue). Women with regular menstrual cycles were studied for one control cycle, 3 consecutive treatment months, and 3-5 months of follow-up. Blood samples were drawn before each monthly dose and at specified time points after the third monthly injection. A total of 16 women were enrolled, 14 of whom completed the study. These 14 women (13 white, one black) ranged in age from 28 to 43.4 years, in body weight from 47.6 to 68.9 kg, and in height from 150 to 175 cm. Mean serum MPA concentrations peaked in the first week after administration of MPA/E2C (Lunelle Monthly Contraceptive Injection). The mean MPA Cmax and AUC0-t(last) were 1.25 ng/mL and 32.13 ng.day/mL, respectively. Serum MPA concentrations declined with a mean terminal half-life of 14.7 days, indicating that absorption from the injection site is prolonged after administration of MPA/E2C. The time for MPA concentrations to fall below the lower limit of quantitation (i.e., < 10 pg/mL) after the third injection ranged from 63 to 84 days. The average MPA trough (Cmin' day 28) concentrations for the three consecutive monthly injections ranged from 0.44 to 0.47 pg/mL, indicating that steady-state conditions were achieved after the first injection. The MPA Cmin values were well above threshold levels required to suppress ovulation throughout the injection interval. Absorption of E2 from the injection site was also prolonged after injection of MPA/E2C. Mean concentrations of E2 peaked at approximately 2 days after the third injection, and the average Cmax was 247 pg/mL. Serum E2 levels declined with a terminal half-life of approximately 8 days; E2 levels returned to baseline (typically, approximately 100 pg/mL) by 14 days after each injection. The average trough (Cmin' day 28) levels for E2 ranged from 40 to 55 pg/mL. The results of this study demonstrate that steady-state conditions are achieved after the first injection of MPA/E2C; no further MPA or E2 accumulation occurs beyond the first injection. Furthermore, the E2 peak observed after injection of MPA/E2C is similar to the nontreated preovulatory E2 range and returns to baseline levels by approximately 14 days after injection.

PIP: This paper characterized the pharmacokinetics and pharmacodynamics of medroxyprogesterone acetate (MPA) and estradiol cypionate (E2C) in 14 healthy, surgically sterile women. These women ranged in age from 28 to 43.4 years, in body weight from 47.6 to 68.9 kg, and in height from 150 to 175 cm. The results of this study demonstrate that steady-state conditions are achieved after the first injection of MPA/E2C; no further MPA or E2C accumulation occurs beyond the first injection. Moreover, the E2 peak observed after injection of MPA/E2C is similar to the nontreated preovulatory E2 range and returns to baseline levels by approximately 14 days after insertion.

Validation of the simultaneous determination of methylprednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic procedure was developed that accurately quantitates methylprednisolone (MP) and methylprednisolone acetate (MPA) in human plasma over the range 2.00-50.0 ng/ml. The internal standard, fluorometholone, was added to an aliquot of sodium fluoride-potassium oxalate-derived plasma. Samples were prewashed with hexane and extracted twice with methylene chloride. The extracts were dried with anhydrous sodium sulfate, centrifuged, and the organic layer separated and dried under nitrogen. The samples were reconstituted in mobile phase and washed an additional time with hexane before 100 microliters were injected onto a Beckman/Altex Ultrasphere ODS column with ultraviolet absorbance detection at 254 nm. Composition of the mobile phase was acetonitrile-water-glacial acetic acid (33:62:5, v/v/v). Calibration curves were obtained by unweighted, linear regression of peak-height ratios of MP (or MPA)/internal standard versus theoretical concentrations of MP or MPA using a Hewlett-Packard 3357 Laboratory Automation System. Extraction efficiencies for MP and MPA over the linear range were 86.4 and 84.7%, respectively. This method was successfully implemented for the analysis of specimens generated from a single-dose bioavailability and safety study for a new formulation of Depo-Medrol sterile aqueous suspension.

Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene.

Recent studies have shown that the 21-kilodalton protein (p21) Ha-ras gene product shares sequence homology with and may exhibit biochemical properties similar to the mammalian guanine nucleotide-binding proteins. These data suggested that one of the biochemical functions of p21 in the vertebrate cell may be to regulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. We determined both in intact NIH-3T3 murine cells and in membranes isolated from these cells that the hormone-stimulated adenylate cyclase activity of cells expressing the EJ human bladder carcinoma oncogene (EJ-ras) is significantly reduced compared with control cells. Thus, the levels of cAMP measured in the EJ-ras-transformed cells by radioimmunoassay are reduced 78% and 93% after prostaglandin and isoproterenol stimulation, respectively, compared with the levels in control cells. Treatment of the EJ-ras-transformed cells with pertussis toxin or cholera toxin did not correct the alterations in adenylate cyclase activity. Cells expressing the normal human Ha-ras gene displayed intermediate levels of adenylate cyclase hormone sensitivity; these levels of adenylate cyclase activity were greater than those in the EJ-ras-transformed cells but lower than in control cells. Hormone-stimulated adenylate cyclase activities in cells transfected with Rous sarcoma virus DNA were similar to those in control cells. These data support the hypothesis that both the normal and mutated Ha-ras p21s are related to guanine nucleotide-binding proteins.

Evidence for 15-HETE synthesis by human umbilical vein endothelial cells.

Incubation of cultured human umbilical vein endothelial cells with [1-14C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF1 alpha. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 15-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 15-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8,15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggest that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4.

Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells.

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer.

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.

Acetylglycerylether phosphorylcholine-(AGEPC) and leukotriene B4-stimulated cyclic AMP levels in human polymorphonuclear leukocytes.

It seems paradoxical that AGEPC induces a transient rise in cyclic AMP, yet the preincubation of neutrophils with agents that elevate cyclic AMP actually inhibits AGEPC-induced aggregation. However, similar transient elevations in cyclic AMP are observed using other stimulators of PMN function such as fmet-leu-phe, C5a (22), immune complexes (24), and phagocytosable particles (11). Elevations in cyclic AMP by PGE1, PGI2, dibutyryl cyclic AMP, and phosphodiesterase inhibitors, before the addition of an agonist, also blocked subsequent neutrophil activation in the above studies. Thus, these observations are not unique to AGEPC. However, the finding that the cyclooxygenase inhibitor indomethacin enhanced AGEPC-stimulated cyclic AMP accumulation is a novel observation and suggests that some oxygenated derivative of the 5-lipoxygenase pathway is responsible for the increase in cyclic AMP. The evidence for the association of the spike in cyclic AMP and the 5-lipoxygenase is strengthened by the observation that the 5-lipoxygenase inhibitor U-60257 attenuates the AGEPC-induced spike in cyclic AMP. It should be noted that U-60257 does not antagonize LTB4-stimulated cyclic AMP accumulation and has no direct influence on the neutrophil adenylate cyclase. The final correlation of the spike in cyclic AMP and the 5-lipoxygenase is made by the fact that LTB4 itself stimulates cyclic AMP levels in intact neutrophils as well as the adenylate cyclase in cell homogenates. As is the case with AGEPC, the transient spike in cyclic AMP induced by LTB4 is coincident with the onset of neutrophil aggregation. However, it is clear that the spike in neutrophil cyclic AMP induced by AGEPC can be dissociated from neutrophil aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist specific desensitization of leukotriene C4-stimulated PGI2 biosynthesis in human endothelial cells.

Leukotriene C4 (LTC4) and, to a lesser extent, leukotriene D4 (LTD4) concentration dependently stimulate prostacyclin (PGI2) biosynthesis in cultured human umbilical vein endothelial cells. PGI2 biosynthesis was quantitated by radioimmunoassay and its structure confirmed by gas chromatography/mass spectrometry. Preincubation of endothelial cells with LTC4 resulted in desensitization to subsequent LTC4 stimulation. However, PGI2 biosynthesis in response to thrombin, PGH2 and arachidonic acid was not inhibited by preincubation with LTC4. The C-6-sulfidopeptide leukotriene receptor level antagonist FPL-55712 attenuates LTC4, but not thrombin-stimulated PGI2 biosynthesis. These data suggest that human umbilical vein endothelial cells have a C-6-sulfidopeptide leukotriene receptor, and that stimulation of this receptor results in PGI2 biosynthesis.

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3',5'-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4.

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.

Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis and cyclic AMP accumulation in human polymorphonuclear leukocytes.

Both LTB4 and AGEPC stimulate PMN aggregation in a concentration-dependent manner. The lipoxygenase inhibitors ETYA and NDGA block AGEPC-induced but not LTB4-induced PMN aggregation. Agents that elevate PMN cyclic AMP levels block both LTB4-and AGEPC-induced aggregation. Paradoxically, shortly after aggregation is initiated by either AGEPC or LTB4, a transient spike in cyclic AMP levels is observed. Subsequent experiments show that LTB4 can directly elevate cyclic AMP in PMNs. Reverse-phase high-pressure liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize LTB4. These data suggest that AGEPC-induced neutrophil aggregation is mediated by LTB4.

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2).

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.

Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.