PGE2 maintains the tone of the guinea pig trachea through a balance between activation of contractile EP1 receptors and relaxant EP2 receptors.

BACKGROUND AND PURPOSE: The guinea pig trachea (GPT) is commonly used in airway pharmacology. The aim of this study was to define the expression and function of EP receptors for PGE(2) in GPT as there has been ambiguity concerning their role. EXPERIMENTAL APPROACH: Expression of mRNA for EP receptors and key enzymes in the PGE(2) pathway were assessed by real-time PCR using species-specific primers. Functional studies of GPT were performed in tissue organ baths. KEY RESULTS: Expression of mRNA for the four EP receptors was found in airway smooth muscle. PGE(2) displayed a bell-shaped concentration-response curve, where the initial contraction was inhibited by the EP(1) receptor antagonist ONO-8130 and the subsequent relaxation by the EP(2) receptor antagonist PF-04418948. Neither EP(3) (ONO-AE5-599) nor EP(4) (ONO-AE3-208) selective receptor antagonists affected the response to PGE(2). Expression of COX-2 was greater than COX-1 in GPT, and the spontaneous tone was most effectively abolished by selective COX-2 inhibitors. Furthermore, ONO-8130 and a specific PGE(2) antibody eliminated the spontaneous tone, whereas the EP(2) antagonist PF-04418948 increased it. Antagonists of other prostanoid receptors had no effect on basal tension. The relaxant EP(2) response to PGE(2) was maintained after long-term culture, whereas the contractile EP(1) response showed homologous desensitization to PGE(2), which was prevented by COX-inhibitors. CONCLUSIONS AND IMPLICATIONS: Endogenous PGE(2), synthesized predominantly by COX-2, maintains the spontaneous tone of GPT by a balance between contractile EP(1) receptors and relaxant EP(2) receptors. The model may be used to study interactions between EP receptors.

Immunoaffinity resin for purification of urinary leukotriene E4.

Urinary leukotriene E4 (LTE4) has been used as an index of total leukotriene synthesis. A wide variety of methods have been applied to measure LTE4 which has made direct comparison of urinary levels reported by different laboratories difficult. A new peptidoleukotriene immunoaffinity resin was utilized for urinary LTE4 purification in a method that is easy and inexpensive, utilizing commercially available reagents. This method is described and compared to other methods. LTE4 (50-250 pg/mL) added to a urine extract was quantitatively recovered using the immunoaffinity resin. Similarly, LTE4 (50-400 pg/mL) added to urine was recovered between 63 and 76%. The coefficient of variation of samples purified and quantified on the same or on different days ranged from 8-10%. There was a strong correlation (r2 = 0.95) between LTE4 concentrations determined after immunofiltration and immunoaffinity purification. Although there was a good correlation between urinary LTE4 levels measured without purification compared to after immunoaffinity purification, the high y-intercept of 179 indicates the presence of interfering substances in unpurified urine. Urinary LTE4 in normal healthy adults was 80 +/- 7 pg/mg creatinine, similar to that previously reported following HPLC or immunofiltration purification. Urinary LTE4 was also measured in healthy children (age 3-12) and found to be 103 +/- 9.

Purification of class I medullipins from the venous effluent of isolated normal kidneys perfused under high pressure with saline.

Medullipin I (Med I) is a vasodepressor prohormone which is continuously elaborated into the renal venous effluent (RVE) of isolated rat kidneys perfused under high pressure. We have improved the yield of Med I by substituting saline for the albumin perfusate previously reported; and considerably improved refinement by directly fractionating the crude lipid extract of the RVE with high pressure liquid chromatography. The results show that Med I, as defined by previous physiologic and pharmacologic criteria, is not a single molecule. The 3 Class I medullipins described here are distinguished by subtle or overt differences in polarity and biologic activity.

A quantitative solid-phase enzymeimmunoassay for 13,14-dihydro-15-keto- prostaglandin F2 alpha in plasma.

Enzymeimmunoassays (EIA) can be viable alternatives to radioimmunoassays (RIA). Indeed, from an environmental perspective, EIA are preferable to RIA. Therefore, the purpose of this project was to develop a quantitative EIA for 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) in bovine plasma. Acetylcholine esterase bound covalently to PGFM, rabbit anti-PGFM, mouse monoclonal anti-rabbit IgG, and PGFM were the principle reagents used for the EIA. Validation experiments indicated that: 1) PGFM standard curves, with doses ranging from 391 to 200,000 fg per microtiter well, were linear; 2) assay sensitivity averaged 391 fg per well; 3) for satisfactory results, PGFM had to be extracted from plasma; 4) content of PGFM in ethyl ether extracts of aliquots from serial dilutions of whole plasma with unknown amounts of PGFM and charcoal-stripped plasma supplemented with known amounts of PGFM did not deviate from parallelism with PGFM standard curves in buffer; 5) correlation between EIA and RIA measurements of PGFM in the same plasma samples was .95; 6) the regression of EIA data on RIA data was linear (Y = .93 x + 83.9; r2 = .91); 7) intra- and interassay coefficients of variation were 3.3 and 10.6%, respectively. The EIA developed in this project is a valid and reliable method for quantitating PGFM in extracts of bovine plasma.

Synthesis and biological activity of 9-deoxo-9-methylene and related prostaglandins.

A number of PGE analogs have been synthesized in which the C-9 carbonyl group has been replaced by an exo-methylene group. These chemically stable 9-deoxo-9-methylene-PGEs exhibit biological profiles very similar to their less stable PGE relatives. 9-Deoxo-16,16-dimethyl-9-methylene-PGE2 (7) retains the useful uterine-stimulating potency of 16,16-dimethyl-PGE2 but is approximately 300 times less enteropooling in the rat. In preliminary clinical trials, 7 has shown efficacy for pregnancy termination by the oral and vaginal routes of administration, as well as relative freedom from gastrointestinal side effects.