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Reporter gene based functional assays for prostanoid receptors: The aequorin luminescence assay
196
EFA & Eicosanoids 1997 - Edinburgh
Concurrent Sessions Tuesday 22 July
T16
T17
Docosahexaenoic Acid plus Arachidonic Acid Enhance Preterm Infant Growth. J Hansen~, D Schadel,C Harris1, K MerkeP, D Adamkin 2, R HalP, M Lim4, F Moya5, D Stevens6, P Twisf. ~/'~dJohr~on(o, ~mviUt, Ill, 2UnivLouilville,louilYille,kl',3(hildremHe~ H~p,Kamal(it'/, HI), "PomaValk,'/I~0,Po~na,CL5$o~y~t~rflHed(tr, gallaL~ %nivSD,lio~ h$, SP,7HinthropUniyflmp,Hin~la, ~, USA
Fish Oil In Pregnancy And Infant Visual Acuity: A Randomized Controlled Trial Olsen SE, Jor~ensen MH, Michaelsan KF, Secher NJ and
Docosahexaenoic acid (DHA} supplementation is reported to enhance visual acuity (VA) of preterm infants at 2 or 4 months (too) corrected age. However, some studies show decreased growth in supplemented infants. Purpose:Wetested the hypotheses that supplementation of premature formulas with algal DHA and funga[ arachidonie acid (ARA} followed by routine term formula feeding beginning near hospital discharge would result in 1) no growth inhibition and 2)enhancedvisualacuity. Methods:In a double-blind conbolledtrial, 194 infants (birth weight: 846-1560g) were randomized to 1 of 3 preterm formulas differing only in fatty acid content: no DHA or ARA (C), 0.15 energy% DHA (D), or 0.14 energy% DHA + 0.27 energy% ARA iDA). Also, a reference group (H) of 90 breast-fed term infants was enrolled. Test formulas were fed for about 28 days before switching to routine term formula. Growth was measured weekly during test formula feeding and at term, 2 mo and 4 mo corrected age (CA). Teller Acui~ Card testing of vtsual acuity was done at 2 and 4 mo CA. Results."Mean + s.c. Growth, g/d ............ Weight, kg ............. VA, cycles/degree(_+oetave) preterm term CA 2 m o C A 4 m e C A 2moCA 4meCA C 30.7_+ 1.1 3.08+0.07 4.71_+0.09 6.05_+0.14 1.72(_+0.10) 3.47(_+0.08) D 33.2+ 1.1 3.05-+0.07 4.66+0.10 5.99+0.14 1.80(+0.10) 3.37(_+0.08) DA 34.7_+ 1.1 3.20+0.06 5.04+0.09 6.31+0.13 1.72(_+0.09) 3.06(_+0.07) H 3.44_+0.06 5.18_+0.09 6.41_+0.13 1.75(_+0.09) 3.85(-+0.07) Neither D nor DA was less than C in infant growth (p=0.966, 0.998) or infant weight at term, 2 mo or 4 mo CA. D and DA did not differ from C in infant visual acuity at 2 (p=0.95) or 4 (p=0.697, 0.071) mo CA. Post hoe analysis shewed DA>C in infant growth (p=0.004) and in infant weight at2 mo CA (p=0.011). In fact, DA did not differ ffom H in infant weight at 2 or 4 mo CA (p=0.23, 0.56) while C and D infant weight remained less than in H at 2 mo (p<0.001) and 4 mo (p=0.028, 0.009). There were no differences in adverse events among formula groups. Conclusions:Supplementationof premature formulas with algal DHA and fungal ARA in amounts comparable to human milk is safe as measured by growth and absence of adverse events. Visual acuity in groups consuming D or DA was not better than C, perhaps because measurements were made more than 2 mo after stopping supplemented formulas which were fed for only abeut 1 too. Notonlywas the previously reported growth suppression not seen with the inclusion of ARA in DA, but enhancement appeared to be achieved compared to C. In conkast to the weight of DA infants which caught up to the reference standard H infants by 2 mo CA, weight with feeding C or D remained less than with H.
Earlier controlled trials have exhibited improved visual acuity in very preterm infants after supplementation ex utero with DHA from a post-conceptional age of about 7 months. We testet whether similar effects could be seen in infants of mothers supplemented with DHA in pregnancy during a comparable post-conceptional period but limited to when the infants were still in utero.
T18
T19
Prostaglandin Metabolism In Developing Human Lung Christine E. C o n n e rI, Rodney W. Kelly,2 Robert H u m e .1 1 University of Dundee, Dundee, Scotland. 2Centre for Reproductive Biology, Edinburgh, Scotland.
Reporter Gene Based Functional Assays For Prostanoid Receptors: The Aequorin Luminescence Assay. Mark Abramovitz, Mark D Ungrin, *Yves Durocher, Rino Stocco, Laila M R Singh, Dean E Sas, Nicole Sawyer and Kathleen M Metters Department of Biochemistry and Molecular Biology, Merck Frusst Centre for Therapeutic Res., P.O. Box 1005, Pointe ClaireDorval, Quebec H9R 4P8 and *Animal Cell Engineering, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2
Prostaglandin PGE2 is a key regulator of human fetal lung development. In human fetal lung organ culture, PGE2 accelerates differentiation of the epithelium to type I and II pneumatocytes including surfactant production, and increases dilatation of the terminal airsacs. The human fetal trachea terminally differentiates early in gestation to a muco-ciliary epithelium but this process is markedly delayed in fetal lung and we propose differential metabolism of PGs is responsible for this asynchronous development. In media from human fetal tracheal and lung organ cultures, levels of PGE2- MOX, PGEM-MOX, PGFM-MOX, TXB2-MOX, 6-keto PGFla-MOX and PGF2ct were determined by radioimmunoassay. PGEM and 15-hydroxyprostaglandin dehydrogenase were localised in fetal lung and trachea by immunohistochemistry-image analysis. 15-hydroxyprostaglandin dehydrogenase mRNA was localised by in-situ hybridisation. In trachea, the predominant PGs are 6-keto PGFlc~, PGFlct and PGE2, but markedly different from fetal lung where PGFM is the major PG with lesser amounts of PGF2c~ and 6-keto PGFlc~. 15hydroxyprostaglandin dehydrogenase and PGFM are predominantly localised to the epithelium of distal lung buds with a more uniform distribution within the epithelium of the fetal trachea. The profile of PGs produced in the developing tracheobronchial system reflects the degree of phenotypic and physiological maturation. In terminally differentiated fetal trachea, the profile of PGs released is similar to that of adult trachea and lung. In contrast, in fetal lung buds, focal catabolism of PGs is predominant and this acts as a regulatory mechanism to retard the process of terminal differentiation of the epithelium.
FOTIP* from Danish Epidemiology Science Centre (Copenhagen), Human Nutrition Research Institute, and Perinatal Epidemiology Research Unit; DENMARK
As part of a large multicentre study*, women were randomized to receive capsules containing 1 g DHA (4 g Pikasol fish oil) or 0 DHA (4 g olive oil) until delivery. Among 132 infants of Danish mothers randomized to one these regimens, 78 infants were tested for visual acuity at postnatal ages 6 to 10 months. Visual acuity was assessed by SWEEP-VEP (swept visual evoked potentials) [logl0(30/cycles per degree)]. SWEEP-VEP fell with infant age (correlation coefficient 0.48, p < 0.001). In the groups having received fish oil and olive oil, mean SWEEP-VEP was 0.248 (SE=0.014, n=39) and 0.258 (SE 0.014, u=39) respectively (p = 0.6). Analyses accounting for infant age, breasfeeding, essential fatty acid composition of milk, and birth outcome will be presented. In this study no effect could be detected of fish oil intake in pregnancy on infant visual acuity. To our knowledge this is the first time this has been studied within the framework of a randomized controlled trial. *Fish Oil Trial In Pregnancy (FOTIP) is a European multicentre randomized controlled trial testing for health effects of fish oil supplementation in pregnancy
We have employed reporter gene based functional assays in order to study prostanoid receptors, members of the G-protein coupled receptor (GPCR) superfamily. Eight prostanoid receptors have been recently cloned and characterized, three of which couple to mobilization of intracellular calcium ([Ca2+]i) (EP1, FP and TP), four of which couple to an increase in intracellular cAMP accumulation (EP2, EP4, DP and IP) and the EP3 receptor which couples to a decrease in intracellular cAMP accumulation. We have established a reporter gene based functional assay which employs aequorin generated luminescence to detect prostanoid receptor elicited [Ca2+]i transients. In order toset up the aequorin luminescence assay, human embryonic kidney (HEK) 293 cells which stably express cytosolic apo-aequorin (AEQ17-HEK 293) have been used as the parental cell line from which double stable EP1, FP or TP prostanoid/aequorin clonal cell lines have been selected. A Labsystems luminoskan luminometer, which accepts 96-well plates, has been set up to run the assay. All three prostanoid receptors were found to couple efficiently and the rank order potency ofprostanoids and selected ligands was determined for each receptor. The effects of antagonists, such as the TP receptor selective SQ29548, can also be readily evaluated using this assay. We have also examined the role that extraeellular Ca2+ plays in EP1, FP and TP receptor mediated signal transduction. The aequorin luminescence assay is not limited to only stably expressing celt lines, transiently expressed receptors in AEQ 17-HEK 293 cells or receptors coexpressed with apoaequorin in other cell lines can be studied as well. In summary, the aequorin luminescence assay is well suited to the study of prostanoid receptors and indeed any GPCR which can couple to the mobilization of [Ca2+]i.
EFA & Eicosanoids 1997 - Edinburgh
Concurrent Sessions Tuesday 22 July
T16
T17
Docosahexaenoic Acid plus Arachidonic Acid Enhance Preterm Infant Growth. J Hansen~, D Schadel,C Harris1, K MerkeP, D Adamkin 2, R HalP, M Lim4, F Moya5, D Stevens6, P Twisf. ~/'~dJohr~on(o, ~mviUt, Ill, 2UnivLouilville,louilYille,kl',3(hildremHe~ H~p,Kamal(it'/, HI), "PomaValk,'/I~0,Po~na,CL5$o~y~t~rflHed(tr, gallaL~ %nivSD,lio~ h$, SP,7HinthropUniyflmp,Hin~la, ~, USA
Fish Oil In Pregnancy And Infant Visual Acuity: A Randomized Controlled Trial Olsen SE, Jor~ensen MH, Michaelsan KF, Secher NJ and
Docosahexaenoic acid (DHA} supplementation is reported to enhance visual acuity (VA) of preterm infants at 2 or 4 months (too) corrected age. However, some studies show decreased growth in supplemented infants. Purpose:Wetested the hypotheses that supplementation of premature formulas with algal DHA and funga[ arachidonie acid (ARA} followed by routine term formula feeding beginning near hospital discharge would result in 1) no growth inhibition and 2)enhancedvisualacuity. Methods:In a double-blind conbolledtrial, 194 infants (birth weight: 846-1560g) were randomized to 1 of 3 preterm formulas differing only in fatty acid content: no DHA or ARA (C), 0.15 energy% DHA (D), or 0.14 energy% DHA + 0.27 energy% ARA iDA). Also, a reference group (H) of 90 breast-fed term infants was enrolled. Test formulas were fed for about 28 days before switching to routine term formula. Growth was measured weekly during test formula feeding and at term, 2 mo and 4 mo corrected age (CA). Teller Acui~ Card testing of vtsual acuity was done at 2 and 4 mo CA. Results."Mean + s.c. Growth, g/d ............ Weight, kg ............. VA, cycles/degree(_+oetave) preterm term CA 2 m o C A 4 m e C A 2moCA 4meCA C 30.7_+ 1.1 3.08+0.07 4.71_+0.09 6.05_+0.14 1.72(_+0.10) 3.47(_+0.08) D 33.2+ 1.1 3.05-+0.07 4.66+0.10 5.99+0.14 1.80(+0.10) 3.37(_+0.08) DA 34.7_+ 1.1 3.20+0.06 5.04+0.09 6.31+0.13 1.72(_+0.09) 3.06(_+0.07) H 3.44_+0.06 5.18_+0.09 6.41_+0.13 1.75(_+0.09) 3.85(-+0.07) Neither D nor DA was less than C in infant growth (p=0.966, 0.998) or infant weight at term, 2 mo or 4 mo CA. D and DA did not differ from C in infant visual acuity at 2 (p=0.95) or 4 (p=0.697, 0.071) mo CA. Post hoe analysis shewed DA>C in infant growth (p=0.004) and in infant weight at2 mo CA (p=0.011). In fact, DA did not differ ffom H in infant weight at 2 or 4 mo CA (p=0.23, 0.56) while C and D infant weight remained less than in H at 2 mo (p<0.001) and 4 mo (p=0.028, 0.009). There were no differences in adverse events among formula groups. Conclusions:Supplementationof premature formulas with algal DHA and fungal ARA in amounts comparable to human milk is safe as measured by growth and absence of adverse events. Visual acuity in groups consuming D or DA was not better than C, perhaps because measurements were made more than 2 mo after stopping supplemented formulas which were fed for only abeut 1 too. Notonlywas the previously reported growth suppression not seen with the inclusion of ARA in DA, but enhancement appeared to be achieved compared to C. In conkast to the weight of DA infants which caught up to the reference standard H infants by 2 mo CA, weight with feeding C or D remained less than with H.
Earlier controlled trials have exhibited improved visual acuity in very preterm infants after supplementation ex utero with DHA from a post-conceptional age of about 7 months. We testet whether similar effects could be seen in infants of mothers supplemented with DHA in pregnancy during a comparable post-conceptional period but limited to when the infants were still in utero.
T18
T19
Prostaglandin Metabolism In Developing Human Lung Christine E. C o n n e rI, Rodney W. Kelly,2 Robert H u m e .1 1 University of Dundee, Dundee, Scotland. 2Centre for Reproductive Biology, Edinburgh, Scotland.
Reporter Gene Based Functional Assays For Prostanoid Receptors: The Aequorin Luminescence Assay. Mark Abramovitz, Mark D Ungrin, *Yves Durocher, Rino Stocco, Laila M R Singh, Dean E Sas, Nicole Sawyer and Kathleen M Metters Department of Biochemistry and Molecular Biology, Merck Frusst Centre for Therapeutic Res., P.O. Box 1005, Pointe ClaireDorval, Quebec H9R 4P8 and *Animal Cell Engineering, Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2
Prostaglandin PGE2 is a key regulator of human fetal lung development. In human fetal lung organ culture, PGE2 accelerates differentiation of the epithelium to type I and II pneumatocytes including surfactant production, and increases dilatation of the terminal airsacs. The human fetal trachea terminally differentiates early in gestation to a muco-ciliary epithelium but this process is markedly delayed in fetal lung and we propose differential metabolism of PGs is responsible for this asynchronous development. In media from human fetal tracheal and lung organ cultures, levels of PGE2- MOX, PGEM-MOX, PGFM-MOX, TXB2-MOX, 6-keto PGFla-MOX and PGF2ct were determined by radioimmunoassay. PGEM and 15-hydroxyprostaglandin dehydrogenase were localised in fetal lung and trachea by immunohistochemistry-image analysis. 15-hydroxyprostaglandin dehydrogenase mRNA was localised by in-situ hybridisation. In trachea, the predominant PGs are 6-keto PGFlc~, PGFlct and PGE2, but markedly different from fetal lung where PGFM is the major PG with lesser amounts of PGF2c~ and 6-keto PGFlc~. 15hydroxyprostaglandin dehydrogenase and PGFM are predominantly localised to the epithelium of distal lung buds with a more uniform distribution within the epithelium of the fetal trachea. The profile of PGs produced in the developing tracheobronchial system reflects the degree of phenotypic and physiological maturation. In terminally differentiated fetal trachea, the profile of PGs released is similar to that of adult trachea and lung. In contrast, in fetal lung buds, focal catabolism of PGs is predominant and this acts as a regulatory mechanism to retard the process of terminal differentiation of the epithelium.
FOTIP* from Danish Epidemiology Science Centre (Copenhagen), Human Nutrition Research Institute, and Perinatal Epidemiology Research Unit; DENMARK
As part of a large multicentre study*, women were randomized to receive capsules containing 1 g DHA (4 g Pikasol fish oil) or 0 DHA (4 g olive oil) until delivery. Among 132 infants of Danish mothers randomized to one these regimens, 78 infants were tested for visual acuity at postnatal ages 6 to 10 months. Visual acuity was assessed by SWEEP-VEP (swept visual evoked potentials) [logl0(30/cycles per degree)]. SWEEP-VEP fell with infant age (correlation coefficient 0.48, p < 0.001). In the groups having received fish oil and olive oil, mean SWEEP-VEP was 0.248 (SE=0.014, n=39) and 0.258 (SE 0.014, u=39) respectively (p = 0.6). Analyses accounting for infant age, breasfeeding, essential fatty acid composition of milk, and birth outcome will be presented. In this study no effect could be detected of fish oil intake in pregnancy on infant visual acuity. To our knowledge this is the first time this has been studied within the framework of a randomized controlled trial. *Fish Oil Trial In Pregnancy (FOTIP) is a European multicentre randomized controlled trial testing for health effects of fish oil supplementation in pregnancy
We have employed reporter gene based functional assays in order to study prostanoid receptors, members of the G-protein coupled receptor (GPCR) superfamily. Eight prostanoid receptors have been recently cloned and characterized, three of which couple to mobilization of intracellular calcium ([Ca2+]i) (EP1, FP and TP), four of which couple to an increase in intracellular cAMP accumulation (EP2, EP4, DP and IP) and the EP3 receptor which couples to a decrease in intracellular cAMP accumulation. We have established a reporter gene based functional assay which employs aequorin generated luminescence to detect prostanoid receptor elicited [Ca2+]i transients. In order toset up the aequorin luminescence assay, human embryonic kidney (HEK) 293 cells which stably express cytosolic apo-aequorin (AEQ17-HEK 293) have been used as the parental cell line from which double stable EP1, FP or TP prostanoid/aequorin clonal cell lines have been selected. A Labsystems luminoskan luminometer, which accepts 96-well plates, has been set up to run the assay. All three prostanoid receptors were found to couple efficiently and the rank order potency ofprostanoids and selected ligands was determined for each receptor. The effects of antagonists, such as the TP receptor selective SQ29548, can also be readily evaluated using this assay. We have also examined the role that extraeellular Ca2+ plays in EP1, FP and TP receptor mediated signal transduction. The aequorin luminescence assay is not limited to only stably expressing celt lines, transiently expressed receptors in AEQ 17-HEK 293 cells or receptors coexpressed with apoaequorin in other cell lines can be studied as well. In summary, the aequorin luminescence assay is well suited to the study of prostanoid receptors and indeed any GPCR which can couple to the mobilization of [Ca2+]i.