M.Helen Maguire

Hypoxia-induced fetoplacental vasoconstriction in perfused human placental cotyledons

Effects of maternal hypoxia on fetoplacental vascular resistance in the human placenta were investigated in an in vitro model in which single anatomic subunits (cotyledons) from term placentas were perfused at constant flow through both fetal and maternal circuits by means of a physiologic salt solution containing dextran. Acute reduction of oxygen tension in the maternal perfusate induced prompt fetoplacental vasoconstriction that recovered rapidly on restoration of oxygen to the perfusate. The response, hypoxic fetoplacental vasoconstriction, could be repeatedly demonstrated in the same cotyledon. The time course of hypoxic fetoplacental vasoconstriction was inversely related to oxygen tension of maternal arterial and maternal and fetal venous perfusates. Maternal and fetal venous perfusate pH and PCO2 did not change during the response. It is concluded that hypoxic fetoplacental vasoconstriction is triggered by decreased oxygen availability. It is suggested that hypoxic fetoplacental vasoconstriction may play a role in local regulation of human fetoplacental blood flow in vivo and may contribute to poor fetal prognosis in preeclampsia.

Determination of adenosine in fetal perfusates of human placental cotyledons using fluorescence derivatization and reversed-phase high-performance liquid chromatography.

A simple, sensitive HPLC method using fluorescence detection was developed for determination of adenosine in fetal venous perfusates of dual-perfused cotyledons from human term placentas. Maternal and fetal circuits of in vitro placental cotyledons were perfused with physiological salt solution containing dextrose and dextran (Earles medium). Conditions were established for optimal formation of fluorescent 1,N6-ethenoadenosine from adenosine and chloroacetaldehyde in Earles medium and for optimal resolution of 1,N6-ethenoadenosine by reversed-phase HPLC of the reaction mixture. The yield of 1,N6-ethenoadenosine was enhanced by dilution and acidification of the sample matrix. Perfusate samples in autosampler vials were diluted 40% with water and reacted with chloroacetaldehyde for 40 min at 100 degrees C; replicate 100-microliters injections were made automatically from each reaction mixture for HPLC analysis with fluorescence detection on a column packed with 3 microns octadecylsilica (Hypersil). Calibration curves were prepared similarly from 4-100 nM adenosine in Earles medium. Alternatively, perfusate samples were diluted twofold with dilute phosphoric acid to give a final pH of 5.4 before reaction with chloroacetaldehyde, and replicate 50-microliters injections were made automatically for HPLC; calibration curves were prepared from 2-400 nM adenosine in Earles medium. 1,N6-Ethenoadenosine was well resolved from Earles-derived artifactual peaks on chromatography with either a linear or a concave gradient of methanol in ammonium phosphate buffer. Total run times were 15 and 19 min, respectively. Sensitivity of measurement of adenosine was 2-4 nM. Derivatization of adenosine using the acidified reaction mixture gave a limit of detection of 100 fmol of adenosine per injection. Application of the method to analysis of adenosine in fetal venous perfusates of eight dual-perfused cotyledons, each from a different placenta, gave a range of 3.5-52 nM adenosine. Ischemia, imposed by cessation of maternal perfusion, caused a two- to sixfold increase in fetal venous perfusate adenosine concomitant with an increase in fetoplacental perfusion pressure; perfusion pressure and perfusate adenosine returned to baseline levels on reperfusion of the maternal circuit. This facile method of determination of perfusate adenosine should allow investigation of the role of placental adenosine release in regulation of fetoplacental vascular resistance and should be applicable to study of adenosine released by other isolated perfused organs.

Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversed-phase high-performance liquid chromatography with photodiode-array detection and on-line validation of peak purity

A new, robust and sensitive reversed-phase high-performance liquid chromatographic method was developed for concomitant measurement of plasma concentrations of the ATP catabolites adenosine and hypoxanthine in human umbilical cord blood. Deproteinized cord plasma was chromatographed on Hypersil C18 columns, using UV photodiode-array detection, spectral analysis of peaks and on-line confirmation of peak purity. Elution with a gradient of acetonitrile-tetrahydrofuran in ammonium dihydrogen phosphate buffer pH 4.7, yielded sharp, well-resolved peaks of adenosine and hypoxanthine within 16 min. Peak areas were quantified from external calibration curves and converted to plasma concentrations via cord blood hematocrits. In seven deliveries, gestational ages 32-40 weeks, adenosine (range, 0.1-2.1 microM) was less than hypoxanthine (range, 1.6-18.5 microM) in the same cord plasma sample. Arteriovenous levels of each purine were similar, except in an abruptio placenta delivery.

Determination of concentrations of adenosine and other purines in human term placenta by reversed-phase high-performance liquid chromatography with photodiode-array detection : evidence for pathways of purine metabolism in the placenta

A robust analytical method, using reversed-phase high-performance liquid chromatography with gradient elution and photodiode-array detection, was used to measure six purines and beta-NAD+ in acid-soluble extracts of samples taken from six different regions of human term placenta. Resolution of the analyte peaks in chromatographic profiles of the extracts, and the use of optimized integration, allowed simultaneous quantitation of all seven analytes from a single chromatogram. Peak purity was confirmed via on-line analysis of peak spectra, utilizing the purity parameter treatment of spectral data. Major placental purines were adenosine, inosine, hypoxanthine and adenine. Except for adenine, concentrations of the purines varied by two-fold or more between different regions of each placenta, but concentration ratios, i.e., adenosine/inosine and inosine/hypoxanthine, were similar. The findings indicate that the pathway of ATP breakdown to hypoxanthine in ischemic human term placenta is via adenosine, and that regional differences in placental concentrations of adenosine and its metabolites may result from regional differences in degree of ischemia.

Measurement of adenosine, inosine and hypoxanthine in human term placenta by reversed-phase high-performance liquid chromatography

An analytical method was developed for measuring adenosine, inosine and hypoxanthine in freshly delivered human term placentas. Representative freeze-clamped samples were taken from the sub-maternal surface of each placenta. Acid-soluble extracts of the samples were analyzed by reversed-phase high-performance liquid chromatography on columns packed with 10-micron porous octadecylsilica, using gradient elution with a linear increase in methanol concentration in ammonium phosphate buffer. Resolution of hypoxanthine from xanthine and adenosine from adenine, and quantitation of hypoxanthine and adenosine were achieved using 0.05 M ammonium dihydrogen phosphate, pH 6.5, as the low-strength eluent. Resolution of inosine from a prominent peak of beta-NAD was optimized using 0.02 M ammonium dihydrogen phosphate, pH 5.6, as low-strength eluent. Recovery of standards was greater than 90%. Mean contents (+/- S.D.) of the analytes in placentas from seven normal deliveries were, adenosine 30.6 +/- 11.5 nmol/g, inosine 68.0 +/- 25.8 nmol/g and hypoxanthine 217 +/- 127.5 nmol/g.

Purinergic Receptors in Visceral Smooth Muscle

Fifty-odd years have elapsed since the first observation of the response of visceral smooth muscle to an adenine nucleotide was reported by Drury and Szent Gyorgi (1929). It is now known that purinergic receptors mediating responses to adenosine and the adenine nucleotides (AMP, ADP and ATP) are present in all types of visceral smooth muscle. Adenine itself and other endogenous purine derivatives appear to have no direct effects, or only minimal effects, on most visceral smooth muscles. Airway smooth muscle is an exception in this regard, and responds to purine bases and non-adenine nucleosides and nucleotides. Knowledge of the distribution of purinergic receptor sites on the plasma membrane of visceral musculature has grown particularly rapidly since Burnstock and his colleagues (1970) proposed that ATP, or a related adenine derivative, is the inhibitory transmitter released from noncholinergic non-adrenergic nerves present in the gut. Much evidence has been presented favoring the view that ATP is the inhibitory transmitter, and evidence to the contrary has also been put forward. The hypothesis remains controversial primarily because specific blockers of the postsynaptic purinergic site, with which the hypothesis might be tested, have not been found. Nevertheless, the numerous studies designed to investigate the purinergic nerve hypothesis have generated much information concerning the nature of the purinergic receptors in visceral smooth muscle.

Evidence for a novel P2X purinoceptor in human placental chorionic surface arteries

To characterize the P2x purinoceptor of arteries of human term placenta, a non-innervated organ, actions of ATP, alpha, beta-methylene-ATP and UTP on de-endothelialized chorionic surface artery segments were compared. ATP and alpha,beta-methylene-ATP caused reversible concentration-dependent contractions, but UTP elicited little or no contraction up to 517 microM. Concentration-effect curves to ATP and alpha,beta-methylene-ATP were parallel, and alpha,beta-methylene-ATP, EC50 4.2 +/- 1.2 microM, was 28-times as potent as ATP. At a saturating concentration, 103 microM, alpha,beta-methylene-ATP did not desensitize the ATP receptor. Contractions to ATP and alpha,beta-methylene-ATP were antagonized by 300 microM suramin. These findings indicate that P2X purinoceptors are present in placental chorionic surface arteries and that they differ from P2X purinoceptors in arteries of other tissues.

Vinca alkaloids inhibit conversion of arachidonic acid to thromboxane by human platelet microsomes: comparison with other microtubule-active drugs

The Vinca alkaloid vinblastine causes dose-dependent inhibition of malondialdehyde formation and aggregation in activated human platelets as a result of inhibition of arachidonic acid metabolism via the thromboxane pathway (Brammer, J.P., Kerecsen, L. and Maguire, M.H. (1982) Eur. J. Pharmacol. 81, 577). The nature of the inhibition by vinblastine has been investigated with human platelet microsomes, measuring conversion of arachidonic acid to malondialdehyde and thromboxane B2 via spectrophotometric assay and RIA, respectively, determining arachidonate oxygenation by monitoring oxygen consumption, and identifying metabolites formed from [1-14C]arachidonic acid. Vinblastine was compared with other Vinca alkaloids and with structurally unrelated microtubule-active drugs. Vinca alkaloids were unique in causing dose-dependent inhibition of both malondialdehyde and thromboxane B2. Order of potency was vinblastine = vincristine = vindesine greater than leurosine greater than vinepidine. Inhibition of malondialdehyde and thromboxane B2 by 50 microM vinblastine was at least 60%. Microsomal cyclooxygenase was not inhibited by 200 microM vinblastine. Inhibition by vinblastine of [1-14C]arachidonic acid conversion to thromboxane B2 was associated with a 4-fold increase in prostaglandin E2 formation. Thromboxane B2, but not malondialdehyde, formation was inhibited by colchicine less than nocodazole much less than vinblastine. Results indicate that microsomal thromboxane synthetase is inhibited by Vinca alkaloids and other tubulin-binding drugs, and suggest that the action of vinblastine in inhibiting thromboxane synthesis, aggregation and release in intact platelets is not dependent upon its antimicrotubular actions.

Effects of vinblastine on malondialdehyde formation, serotonin release and aggregation in human platelets

Effects of the microtubular agent vinblastine on human platelet malondialdehyde formation, [14C]serotonin release and aggregation were studied in suspensions of [14C]serotonin-labelled platelets. Vinblastine caused dose-dependent inhibition of malondialdehyde formation and aggregation in platelet suspensions stimulated with thrombin, ADP or palmitaldehyde acetal phosphatidic acid (PGAP). Malondialdehyde formation, aggregation and [14C]serotonin release caused by threshold doses of thrombin were reduced but not abolished by 100 muM vinblastine; 30-100 muM vinblastine abolished ADP- and PGAP-induced malondialdehyde formation and [14C]serotonin released and transformed ADP- and PGAP-induced irreversible aggregation to a diminished reversible response. Arachidonate conversion to malondialdehyde catalysed by human platelet microsomes was inhibited by vinblastine and the cyclooxygenase inhibitors indomethacin and aspirin, but not by salicylate. Vinblastine inhibited the microsome-catalysed formation of malondialdehyde from prostaglandin H2. It is concluded that vinblastine inhibits the thromboxane pathway of arachidonate metabolism in stimulated platelets, consequently inhibiting release and aggregation, and that this effect of vinblastine may be, at least in part, independent of its antimicrotubular actions.

Measurement of hypoxanthine and xanthine in late-gestation human amniotic fluid by reversed-phase high-performance liquid chromatography with photodiode-array detection

A robust analytical method was developed for measurement of hypoxanthine and xanthine in late-gestation human amniotic fluid by reversed-phase high-performance liquid chromatography using diodearray detection. Purity of analyte peaks was confirmed via on-line analysis of peak spectra utilizing the purity parameter treatment of spectral data. Amniotic fluid obtained by amniocentesis was deproteinized by centrifugal ultrafiltration and chromatographed on an octadecylsilica column using isocratic elution with 1% (v/v) acetonitrile in 0.05 M ammonium dihydrogenphosphate pH 6.0; hypoxanthine and xanthine were resolved, but the hypoxanthine peak was not pure. Chromatography on a column of polar endcapped octadecylsilica, using similar mobile phase conditions, yielded spectrally pure peaks of hypoxanthine xanthine. Hypoxanthine and xanthine levels in amniotic fluid from fourteen patients, gestational age 34-39 weeks, ranged from 0.56 to 2.74 microM and 1.62 to 5.52 microM, respectively.