Denis J. Crankshaw

Synergistic actions of nitrovasodilators and isoprenaline on rat aortic smooth muscle

Previous studies have established that nitrovasodilators potentiate the inhibition of platelet function by activators of adenylyl cyclase, but uncertainty exists as to whether a comparable effect is seen in vascular smooth muscle. We initially studied the effects of the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), on the relaxation by isoprenaline of rat aortic smooth muscle that had been precontracted by phenylephrine. Concentrations of SNP (0.25 nM) and SIN-1 (30 nM) that relaxed aortic smooth muscle less than 30% alone, caused significant (3-fold) decreases in the IC50 values for isoprenaline. The cAMP phosphodiesterase inhibitors, cilostamide (20 nM) and Ro 20-1724 (10 microM), caused comparable reductions in the IC50 values for isoprenaline. At these concentrations, each of the four compounds also increased the maximum relaxation achieved with isoprenaline. Even more marked synergistic interactions were observed between isoprenaline and either the nitrovasodilators or the cAMP phosphodiesterase inhibitors when these compounds were added simultaneously before contraction of aortic smooth muscle by phenylephrine. Thus, concentrations of SNP (5 nM), SIN-1 (1 microM), cilostamide (1 microM) and Ro 20-1724 (100 microM) that inhibited contraction by less than 30% decreased the IC50 values for isoprenaline by 8- to 10-fold. At the above concentrations, these compounds each caused a supra-additive inhibition of contraction when added with 100 nM isoprenaline. Thus, synergism between nitrovasodilators and isoprenaline, an activator of adenylyl cyclase, could be detected in vascular smooth muscle and was particularly marked when inhibition of contraction was studied. This action of nitrovasodilators resembled that of inhibitors of cAMP phosphodiesterase.

A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry.

Heterologous expression of KCNQ2 (Kv7.2) results in the formation of a slowly activating, noninactivating, voltage-gated potassium channel. Using a cell line that stably expresses KCNQ2, we developed a rubidium flux assay to measure the functional activity and pharmacological modulation of this ion channel. Rubidium flux was performed in a 96-well microtiter plate format; rubidium was quantified using an automated atomic absorption spectrometer to enable screening of 1000 data points/day. Cells accumulated rubidium at 37 degrees C in a monoexponential manner with t(1/2)=40min. Treating cells with elevated extracellular potassium caused membrane depolarization and stimulation of rubidium efflux through KCNQ2. The rate of rubidium efflux increased with increasing extracellular potassium: the t(1/2) at 50mM potassium was 5.1 min. Potassium-stimulated efflux was potentiated by the anticonvulsant drug retigabine (EC(50)=0.5 microM). Both potassium-induced and retigabine-facilitated efflux were blocked by TEA (IC(50)s=0.4 and 0.3mM, respectively) and the neurotransmitter release enhancers and putative cognition enhancers linopirdine (IC(50)s=2.3 and 7.1 microM, respectively) and XE991 (IC(50)s=0.3 and 0.9 microM, respectively). Screening a collection of ion channel modulators revealed additional inhibitors including clofilium (IC(50) = 27 microM). These studies extend the pharmacological profile of KCNQ2 and demonstrate the feasibility of using this assay system to rapidly screen for compounds that modulate the function of KCNQ2.

Effects of some isoprostanes on the human umbilical artery in vitro

Cumulative concentration‐effect curves for the selective prostanoid TP receptor agonist U46619 and six isoprostanes were constructed in the human isolated umbilical artery. All compounds except 8‐iso‐PGF3α produced concentration‐dependent contractions. The contractile response to the isoprostanes increased with each cumulative addition up to a point, after which subsequent addition reduced the contraction below the previous level. This [downturn] in the concentration‐effect curve did not occur with U46619. The potencies of the compounds tested were as follows (pEC50±s.e.mean): U46619, 6.7±0.2; 8‐iso‐PGE2, 6.5±0.1; 8‐iso‐PGF2α, 5.8±0.2; 8‐iso‐PGE1, 5.4±0.1; 8‐iso‐PGF1α, 5.0±0.1; 8‐iso‐PGF2β> 4.8; 8‐iso‐PGF3α>> 4.8 (n=4–17). Neither 8‐iso‐PGF2β nor 8‐iso‐PGF3α at 44 μM had a significant effect on cumulative concentration‐effect curves to U46619. The selective TP receptor antagonist GR32191 (0.1 μM) caused rightward shifts in the concentration‐effect curves to all the active compounds. pA2 values for GR32191 against U46619, 8‐iso‐PGE2, 8‐iso‐PGF2α, 8‐iso‐PGE1 were 7.6±0.2, 9±1, 8.2±0.3 and 7.7±0.3, respectively (n=4). Neither Nω‐nitro‐L‐arginine methyl ester (100 μM) nor the selective DP receptor antagonist BW A868C (50 nM) affected the complex concentration‐effect curve to 8‐iso‐PGE2 (n=3). Stable contractions to U46619 (1–3 μM) were unaffected by anandamide at concentrations up to 60 μM.

Receptors for E type prostaglandins in the plasma membrane of nonpregnant human myometrium

Abstract Binding sites for prostaglandin E 1 were present in the 1000 g supernatant of nonpregnant human myometrium. When the 1000 g supernatant was fractionated the distribution of prostaglandin E 1 binding sites followed that of the plasma membrane markers, phosphodiesterase-I and 5′-nucleotidase, but was different from that of the endoplasmic reticulum marker NADPH-cytochrome c reductase or the mitochondrial marker succinatecytochrome c reductase. It is concluded that a major portion of the prostaglandin E 1 receptors in the human myometrium is located at the plasma membrane. Scatchard analysis of prostaglandin E 1 binding to the plasma membrane-enriched fraction indicated the presence of both high and low affinity sites.

The effects of some synthetic prostanoids on the contractility of the human lower uterine segment in vitro

We have investigated the ability of three synthetic prostanoids to directly influence uterine contractility by studying the effects in vitro. Strips of lower uterine segment smooth muscle were obtained from women undergoing elective cesarean section at term. The ability of these strips to develop tension in the presence of cumulative additions of prostanoids or oxytocin was assessed. Spontaneous contractions were inhibited by ZK 96.480, a stable synthetic analog of prostaglandin I2, with a 50th percentile effective concentration (EC50) of 8 nmol/L. Both sulprostone, an analog with selectivity for some of the actions of prostaglandin E2, and U-44069, a stable thromboxane A2 mimetic, caused excitation with EC50s of 20 and 16 nmol/L, respectively. The EC50 for oxytocin was 6 nmol/L. There were no significant differences in the maximal tensions developed in response to the excitatory prostanoids or oxytocin.

Functional characterization of the prostanoid DP receptor in human myometrium

Spontaneous contractile activity of strips of human myometrium obtained from non-pregnant donors at the time of hysterectomy was inhibited by the selective prostanoid DP receptor agonists BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin) and ZK110841 ((5Z,13E)-(9R,11R,15S)-9 beta-chlor-15-cyclohexyl-11,15-dihydroxy-16,17,18,19, 20-pentanor-5,13-prostadienoic acid) with pEC50 values of 8.4 and 7.3 respectively but prostaglandin D2 produced a biphasic effect. In the presence of the TP receptor antagonist L670596 ((-)-6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid), contractile activity induced by the FP receptor agonist, cloprostenol ([1R-[1 alpha(Z),2 beta(1E,3R),3 alpha,5 alpha]]-7-[2-[4-(3- chlorophenoxy)-3-hydroxy-7-butenyl]-3,5-dihydroxycyclopentyl]-5-he ptenoic acid), was inhibited by BW 245C (pEC50 = 7.5), ZK110841 (pEC50 = 6.7) and prostaglandin D2 (pEC50 = 6.3). Under these conditions both prostaglandin J2 and 9 alpha,11 beta-prostaglandin F2 were inhibitory partial agonists. All compounds were antagonized by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)h ydantoin), but the pKB values were both concentration-dependent (pKB versus BW 245C at 10 nM = 9.1, at 50 nM = 8.3) and agonist-dependent (pKB at 10 nM versus BW 245C = 9.1, versus ZK110841 = 7.4). Both agonist and antagonist potencies support the existence of DP receptors in human myometrium. The concentration and agonist dependence of the action of BW A868C suggests that putative DP receptor agonists relax human myometrium by more than one mechanism. These observations may be explained by the existence of subtypes of DP receptor in human myometrium.

Selective cytochrome P450 3A4 inhibitory activity of Amaryllidaceae alkaloids

A library of natural and semi-synthetic Amaryllidaceae alkaloids was screened for cytochrome P450 3A4 (CYP3A4) inhibitory activity. Of the crinane, lycorane and galanthamine representatives examined two semi-synthetic silylated lycorane analogues, accessed via a chemoselective silylation strategy from lycorine, and the natural compound narciclasine exhibited low micromolar activities. Important pharmacological features uncovered include the lack of CYP3A4 inhibitory activity seen for galanthamine and the selective activity that is seen with narciclasine over pancratistatin.

Characterization of excitatory prostanoid receptors in the human umbilical artery in vitro.

5‐HT and the prostanoid TP receptor agonists, U46619 and I‐BOP, constricted the human umbilical artery with pEC50 values of 7.3±0.2, 6.7±0.1, and 7.3±0.2, respectively. The selective TP receptor antagonist, GR32191 (0.1 μM), shifted the concentration‐effect curves to U46619 and I‐BOP to the right, but had no effect on the response to 5‐HT. The natural prostaglandins, PGF2α and PGE2, caused concentration‐dependent contraction with pEC50 values of 5.2±0.2 and 4.9±0.2, respectively. PGD2 was a partial agonist with a pEC50 of 5.24±0.03. GR32191 (0.1 μM) inhibited the responses to all of these compounds suggesting that they produce contraction by acting at TP receptors. Sulprostone failed to elicit contraction in the human umbilical artery at concentrations up to 4.4 μM suggesting the absence of EP1 and EP3 receptors. Despite this, 17‐phenyltrinor PGE2 and GR63799 both induced contraction at concentrations above 1 μM, but the effects were sensitive to GR32191 (0.1 μM). Fluprostenol had no effect on the human umbilical artery at concentrations up to 17 μM suggesting the absence of FP receptors. Cloprostenol was ineffective in two tissues, but caused contraction in one tissue at the highest concentration tested (1.7 μM). However, this response was abolished in the presence of GR32191 (0.1 μM). The effects of four TP receptor antagonists were assessed by global non‐linear regression analysis. GR32191, SQ29548, SQ30741, and ICI192605 competitively inhibited responses to U46619 with pKb values of 8.0±0.1, 7.6±0.1, 7.0±0.2 and 8.1±0.1, respectively. These results suggest that the human umbilical artery functionally expresses TP receptors, but not EP1, EP2 or FP receptors.

Human Cytochrome P450 Liability Studies of trans-Dihydronarciclasine: A Readily Available, Potent, and Selective Cancer Cell Growth Inhibitor

The cytochrome P45O activities of the naturally occurring Amaryllidaceae alkaloid narciclasine (3), isolated from Narcissus pseudonarcissus, and synthetic derivative trans-dihydronarciclasine (5) are reported. While narciclasine was found to possess potent inhibitory activity to human CYP3A4, its dihydro analogue was inactive. This study revealed that the C1-C10b double bond is required for inhibition of this crucial metabolizing enzyme. Compound 5 also demonstrated no inhibition of the related human cytochromes CYP19 and CYP1A1. This study elevates the status of trans-dihydronarciclasine (5) as a highly privileged, readily available molecule, with potent and selective anticancer activity.

Isolation of flavonoids from the heartwood and resin of Prunus avium and some preliminary biological investigations

An investigation of the constituents in heartwood and resin of Prunus avium is reported. A mini-library of structurally diverse flavanones and flavones was screened for human cytochrome P450 1A1, 3A4 and 19 (aromatase) inhibition, and for antifungal activity against a panel of pathogenic fungi. The defensive role of these natural plant flavonoids as antifungal phytoalexins and phytoanticipins is discussed.