Brenda W. Griffin

Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists

Various prostaglandin agonists representing various classes of receptor subtypes were evaluated for their ability to stimulate adenylyl cyclase via the endogenous DP receptor in embryonic bovine tracheal (EBTr) cells. Two antagonists were used to block the agonist‐induced cyclic AMP production. ZK118182 (EC50=16±4 nM), RS‐93520 (EC50=23±4 nM), SQ27986 (EC50=33±9 nM), ZK110841 (EC50=33±5 nM), BW245C (EC50=59±19 nM) and PGD2 (EC50=101±10 nM) (n=4–70) were the most potent agonists. Whilst most compounds were full agonists (Emax=100% relative to PGD2), BW245C was significantly more efficacious than PGD2 (Emax=121±3%; P<0.001) and RS‐93520 appeared to be a partial agonist (Emax=64±9%; P<0.001). Agonists from the EP (e.g. enprostil; misoprostol; butaprost), FP (e.g. cloprostenol; fluprostenol; PHXA85), IP (iloprost; PGI2) and TP (U46619) prostanoid receptor classes were weak agonists or inactive in the EBTr cell assay system. The DP‐receptor antagonist, BWA868C, showed a competitive antagonist profile with pA2 values of 8.00±0.02 and 8.14±0.13 in Schild analyses with two structurally different agonists, BW245C and ZK118182, respectively (n=3). AH6809, another purported DP‐receptor antagonist, weakly inhibited PGD2‐ and ZK118182‐induced cyclic AMP production (Kis=808±193 nM and 782±178 nM, respectively). The current studies have characterized the DP receptor positively coupled to adenylyl cyclase in EBTr cells using a wide range of agonist and antagonist prostaglandins. These data support the utility of the EBTr cell line as a useful tool for the evaluation of DP receptor agonists and antagonists and for profiling other classes of prostaglandins.

Spectra of chloroperoxidase Compounds II and III

Chloroperoxidase was present as Compound II during the peroxidatic oxidation of ascorbic acid. Compound III (oxy-form) was formed when excess hydrogen peroxide was added to Compound II. By decreasing the temperature it was possible to measure the spectra of Compounds II and III in the Soret and visible regions. Each spectrum was found to resemble that of the corresponding form of lactoperoxidase. Under the experimental conditions, chloroperoxidase Compound III was apparently converted to Compound II in parallel with the decomposition of hydrogen peroxide and finally to the ferric enzyme.

Use of a semi-automated, robotic radioimmunoassay to measure cAMP generated by activation of DP-, EP2-, and IP-prostaglandin receptors in human ocular and other cell types

The aim of these studies was to compare the effects of several prostaglandin agonists on adenylyl cyclase activity in embryonic bovine tracheal (EBTr) cells, transformed human nonpigmented ciliary epithelial (NPE) cells and National Cancer Bank (NCB-20) cells. These cell types have been shown to express DP, EP2 and IP prostaglandin (PG) receptors, respectively. Cyclic AMP (cAMP) generation was measured by manual and semi-automated radioimmunoassay (RIA) techniques. ZK118182 (EC50 = 10-27 nM), PGE2 (EC50 = 21-27 nM) and PGI2 (EC50 = 3.5-4 nM) had the highest potency at the DP, EP2 and IP receptors, respectively. A plot of potency (EC50) values generated with both techniques showed a high degree of correlation for all three receptors. These studies provide further characterization of prostanoid receptor functional responses in three cell types and demonstrate the advantages of a semi-automated RIA method for the analysis of the second messenger cAMP.

Characterization of the reduction of 3-acetylpyridine adenine dinucleotide phosphate by benzyl alcohol catalyzed by aldose reductase.

Aldose reductase from rat lens catalyzes reduction of the 3-acetylpyridine analog of NADP+ by benzyl alcohol; the fluorescence of the reduced pyridine nucleotide produced in this reaction forms the basis of a new, more sensitive assay for this enzyme. The pH dependence and kcat/Km ratio of this enzymatic reaction are very similar with NADP+ or the analog; however, at pH 7.5, the reaction with the analog has a sevenfold greater kcat. In an NADPH-supported reduction reaction, aldose reductase exhibited similar activity toward benzaldehyde and glyceraldehyde, but benzyl alcohol was a much better substrate than physiological polyols in the analog-dependent oxidation reaction: relative kcat/Km ratios were 740 for benzyl alcohol, 2.2 for xylitol, and 1.0 for glycerol. By this assay, a new, high-affinity aldose reductase inhibitor AL-1567 showed noncompetitive inhibition with the pyridine nucleotide analog, with Ki = 2.05 X 10(-6) M, and competitive inhibition with benzyl alcohol, with Ki = 5.7 X 10(-9) M, indicating that the inhibitor binds the free enzyme with extremely high affinity. Compared to spectrophotometric assays of aldose reductase activity, the fluorometric assay extends the lower limit of detectability of enzyme activity by a factor of 100, and allows the determination of Ki values for potent inhibitors of the enzyme with greater accuracy.

A New Chromatographic Method for Measurement of Rubidium Transport Activities in Cultured Bovine Retinal Pigment Epithelial Cells

A new method for measuring cellular rubidium (Rb+) uptake activities based on cation chromatography was developed and compared with the standard technique, uptake of the radioisotope 86Rb+, using cultured bovine retinal pigment epithelial (RPE) cells. The Rb+ response was strictly linear from 0.25 nmol (detection limit) to 25 nmol. The Na+/K(+)-ATPase inhibitor ouabain inhibited Rb+ uptake with IC50 values of 128.7 +/- 23.5 nM (n = 8; radioactive method) and 56.6 +/- 9.3 nM (n = 9; non-radioactive method, p < 0.01). The latter value is identical to the IC50 value of 54.4 +/- 16.2 nM (n = 3) for ouabain binding to the intact RPE cells. Ouabain and bumetanide, an inhibitor of the Na+/K+/Cl(-)-cotransporter, each inhibited Rb+ uptake maximally by 66 and 30%, respectively. This new technique allows sensitive measurement of intracellular Rb+, as well as K+ and Na+, and, thus, should prove useful for studying the effects of pharmacologic agents and simulated disease conditions on cation transport and cation balance in RPE and other cell types.

Comparison of the Pharmacokinetics and Pharmacodynamics of the Aldose Reductase Inhibitors, AL03152 (RS), AL03802 (R), and AL03803 (S)

The pharmacokinetics of AL03152 (RS) and its enantiomers, AL03802 (R) and AL03803 (S), were studied in the Sprague–Dawley rat following intravenous bolus administration. The enantiomers had differing pharmacokinetic profiles, while the racemic compound exhibited pharmacokinetic parameters approximating the mean values of the individual enantiomers. The total clearance (CLT) values of the two enantiomers were similar, but the intrinsic clearance (Clint) was much greater for the S-enantiomer than for the R-enantiomer. The volume of distribution (Vss) for AL03802 (R) was threefold greater than that for AL03803 (S). The stereoselectivity in Vss could not be totally accounted for by the slight difference in serum protein binding of the isomers and resulted in a difference in the half-lives of the enantiomers. Only the R-isomer exhibited a persistent terminal elimination phase, consistent with more extensive tissue binding than the S-isomer. AL03152 enantiomers were equivalent in potency assessed from in vitro IC50 values toward rat lens aldose reductase and rat kidney L-hexonate dehydrogenase and lens EC50 values in diabetic rats.

Pharmacokinetics and efficacy of structurally related spirohydantoin and spirosuccinimide aldose reductase inhibitors

1. Six potent aldose reductase inhibitors (ARI), three spirohydantoin (I to III) and three spirosuccinimide (IV to VI) compounds, showed similar IC50 activities in vitro for the inhibition of rat lens aldose reductase, but their ED50 values in diabetic rats varied as much as 20-fold in the lens and 50-fold in the sciatic nerve tissue. Pharmacokinetic studies were undertaken to investigate these findings. Structure-pharmacokinetic relationships were studied following i.v. administration to cynomolgus monkeys. 2. The clearance (CL) of each spirosuccinimide ARI was faster (greater than 5 times) than that of the corresponding spirohydantoin compound. In both series the CL values of the C(4) methyl and methoxy analogues were 4-fold greater than those for the unsubstituted compounds, although the CL values of the methoxy and methyl derivatives in the same series were not significantly different. 3. The volumes of distribution (Vss) of the spirohydantoins were about one-half those of the corresponding spirosuccinimides, and the Vss values of the parent compounds of both ARI series did not differ dramatically from those of their methyl and methoxy analogues. 4. All six compounds were eliminated from plasma in a biexponential fashion. The half-lives (lambda 1 and lambda 2) of the spirohydantoin compounds were much longer than those of the corresponding spirosuccinimide compounds, and the unsubstituted compounds had longer half-lives than their methyl and methoxy derivatives. The longest lambda 1 and lambda 2 half-lives were observed for imirestat, while two of the spirosuccinimides had the shortest half-lives. 5. These results indicate that the relationships observed between the in vitro and in vivo activities of the six ARI can be attributed to structurally dependent differences in metabolic clearance.

Role of Aldose Reductase and Effects of Aldose Reductase Inhibitors in Ocular Tissue Aging Phenomena in the Diabetic Rat

Studies of the role of aldose reductase in sugar cataractogenesis have been aided by the synthesis and characterization of inhibitors of this enzyme which can prevent lens opacification in galactosemic or diabetic rats1,2. The anti-cataract activity of aldose reductase inhibitors correlates with inhibition of polyol accumulation in the lenses of treated galactosemic or diabetic animals; in untreated diabetic rats, lens sorbitol levels may be elevated 100-fold over the normal value3. Hyperglycemia produces an increase in glucose content of tissues not responsive to insulin. Due to increased substrate availability, the rate of sorbitol formation by action of aldose reductase is greatly accelerated relative to conversion of this metabolite to fructose via sorbitol dehydrogenase1:

Pharmacology of [3H]Prostaglandin E1/[3H]Prostaglandin E2 and [3H]Prostaglandin F2 α Binding to EP3 and FP Prostaglandin Receptor Binding Sites in Bovine Corpus Luteum: Characterization and Correlation with Functional Data

Glucose\quad + \quad NADPH\quad + \quad {H^{ + }}\;\xrightarrow{{Aldose\,Reductase}}\;Sorbitol\quad + \quad NAD{P^{ + }}