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Telomerase activity in human trophoblast
A.8
Placenta
TELOMERASE ACTIVITY IN HUMAN TROPHOBLAST. K.Isakaf, H.Nishi’, A.Fujito’, H.Ito’, T.Yudate’, K.Shiraishi’, K.Ohyashiki’ and M.Takayama’. ‘Dept. of Obstetrics and Gynecology and ‘First Dept. of Internal Medicine, Tokyo Medical College, Tokyo, JAPAN
REGULATION OF CL- AND K+ ‘TRANSPORT BY THE HUMAN PLACENTA C.P. Sibley, S.L. Greenwood, J.D Glazier, M.K. Sides and M. Turner, Department of Child Health and School of Biological Sciences, University of Manchester, St Mary’s Hospital, Manchester, Ml3 OJH, UK
Obiective ; Recent reports have suggested that many proliferating normal cells express telomerase activity, although telomerase activity was considered to be specific for cancer cells. These results indicate that telomerase activity is not restricted to cancer cells and that the somatic expression of telomerase is likely to be widespread. It is therefore interesting to investigate telomerase activity in trophoblasts which have characteristics of proliferation, differentiation and invasion. Methods ; Subpopulations of trophoblasts were separated by a modification of Kbman~s method and MACS (magnetic cell sorting) with antibodies of integrins. Telomerase activity was assayed semiquantitatively using fluorescence-based TRAP (modified telometic repeat amplification protocol). A cytological detection of telomerase activity was also examined by newly developed in situ TRAP procedure and the expression of human telomerase reverse transcriptase subunit (hTRT) was detected by RT-PCR. Results; Telomerase activity was detected in 43% of normal placental tissues with a tendency for its activity to decrease in accordance with gestational age. These telomerase activities showed a close correlation with expression of hTRT. In subpopulation of cytotrophoblasts (CTs) separated from early and late placenta, telomerase activity was predominantly detected in column CTs from early placenta. Fluorescence signals after in situ TRAP were observed in the trophoblastic cell layers in early placenta as well as those separated CTs, but not in late placenta. Conclusion ; These results suggest that telomerase activity correlates with the regulation of trophoblastic cell proliferation. We confirmed that in situ TRAP was useful for the detection of telometase positive cells.
By analogy with other tissues it is likely that there is acute regulation of ion transport across the placenta in response to e.g. changes in maternal and fetal plasma volume and in intracellular and extracellular osmotic pressure. Indeed, we have previously reported rapid effects of adrenaline on ion transport across the pig placenta in ~$0 and in vitro. Here we address the question of whether Cl- and K transport by the human placental syncytiotrophoblast can be acutely regulated. As regards C1’, regulatahle transport proteins are present in cytotrophoblast cells and syncytiotrop$blast; whole cell patch clamp studies show the presence of Ca -activated and volumeactivated Cl- currents; the cystic fibrosis transmembrane conductance regulator, a CAMP activated CI’ channel is expressed by the placenta and hCG can activate C1’ currents in the cytotrophoblast cell. Direct evidence of regulation comes from studies in which we fmd that increasing cell volume (induced by lowering extracellular osmolality) and raising [Ca”], (addition of the ionophore A23 187) both increase “CI’ efffux from cytotrophoblast cells pre-loaded with the tracer; interestingly addition of a CAMP ‘cocktail’ had no effect on efflux. As regards K’, we also have pvidence that an increase in cell volume and a rise in [Ca*‘li increase 6Rb (used as a marker of K+) eflux from cytotrophoblast cells and frpents of term placental villi. A variety of hormones increase [Ca Ii of the syncytiotrophoblast, including angiotensin II (AH), ATP and acetylcholine. We have recently found that AIIgnd ATP, but not acetylcholine, cause a dose dependent increase in Rb efflux from vilhts fragments. AI1 induced a maximum increase in effhtx of 18+4% (mean* s.e.; n=S) at 1OnM and caused a peak increase of48*15% at lOurn. ATP caused a peak increase in efflux of 38*8% at IOOuM. These data suggest that there is acute hormonal regulation of C1’ and K’ transport by the human placenta. Such effects are likely to be important in fetal homeostasis. Supported by the Medical Research Council.
NITRIC OXIDE AND OTHER REACTIVE OXYGEN SPECIES REGULATE HUMAN-FETAL PLACENTAL VASCUIAR RESISTANCE. L Mvatt, W. Kosaenjans, A Eis, D. Brockman. Department of Obstetrics and Gynscotogy, Unlvershy of Cincinnati College of Medicine, Cincinnati, OH 45267 USA
THE REGULATION OF MATERNAL PLACENTAL CIRCULATION. A.M. Carter, Department of Physiology, University of Odense, Winslowparken 19, Odense, Denmark.
The nitric oxide radical (NO) maintains low vascular resistance and attenuates the action of vasoconstrictors In the human fetal-placental vascular. The superoxide radical (0;) inactivatea NO but if both 0; and NO are produced in sxcess, they interact to form the long-lived powerful oxidant peroxynftritq ONOO; which can cause vascular dysfunction. Increased formation and action of ON00 vtsuaiized as nitrotyrosine residues is seen In the fetal-placental vasculature of pregnancies complicated by nitrotyrossne preeclampsla, Intrauterine growthrestriction (IUGR) andinsullndspendentdlabetesmayindlcate vascular damage leading toincreased resistance toflow Sean in these pregnancies. To assess the functionality of peroxynitrite action In the fetal-placental vasculature, we measured responses to the vasodilators prostacyctin (PGI,) and the NO donor nttroglycerinq or the vasoconstrictors angiotensin II and the thromboxane mimetic U49519 (all lo-+-19%) in isolated perfused placental cotyledons (n=5 preparations for each treatment) before or following perfuslon with authentic ON00 (10%) for 30 minutes. Following treatment with ONOO’, vasodilator responses to PGI, and nitroprusslde were significantly attenuated (p<0925ANOVA) as was thevascconstrictor response to U45919 (p~O.991 ANOVA). However, the vasoconstrictor response to All was unaffected by ON00 treatment. Attenuation of vasodilator responses to PGI, and the NO donor was greater than the vasoconstrlctor response to U49919, mimicking the altered vasoactive responses reported in preeclampsla These findings suggest that the oxtdatlve stress of preeclampsia, IUGR and diabetic pregnancies leads to production of the powerful oxidant peroxynitrite which can cause endothelial dysfunction/vascular damage leading to altered vascular reactivity and blood flows In the placenta
The human placenta is hemochorial; consequently, it lacks the arterioles that elsewhere regulate vascular resistance and blood flow. Regulation of uteroplacental blood flow, if it occurs, must be at the level of the intramyometrial arteries. Myometrial blood vessels do receive innervation, but the EC,, for noradrenaline is in the uM range and sympathetic vasoconstriction likely does not play a key role. The most potent vasoconstrictors are two hormones, arginine vasopressin and angiotensin II. The intramyometrial arteries are exquisitely sensitive to AVP, with an EC,, of 60-100 pM. However, the concentration of free AVP in plasma is even lower (l-2 PM), being reduced in the course of pregnancy. Plasma concentrations of angiotensin II in normotensive women are in the pM range compared to an EC,, in the nM range, so the renin-angiotensin system is unlikely to participate in the physiological control of uteroplacental blood flow. Intramyometrial arteries are more sensitive to thromboxane and endothelin-I than to noradrenaline, less so than to AVP. Endothelin-1 is an effective constrictor of intramyometrial arteries at nM concentrations. Substance P and vasoactive intestinal peptide are neurotransmitters in the genital tract; both are able to dilate intramyometrial arteries, and substance P is effective at pM doses. There is an endothelium-derived relaxing factor in uterine arteries. In addition, nitric oxide synthase is present in mural trophoblast.
(1998),
Vol. 19
Placenta
TELOMERASE ACTIVITY IN HUMAN TROPHOBLAST. K.Isakaf, H.Nishi’, A.Fujito’, H.Ito’, T.Yudate’, K.Shiraishi’, K.Ohyashiki’ and M.Takayama’. ‘Dept. of Obstetrics and Gynecology and ‘First Dept. of Internal Medicine, Tokyo Medical College, Tokyo, JAPAN
REGULATION OF CL- AND K+ ‘TRANSPORT BY THE HUMAN PLACENTA C.P. Sibley, S.L. Greenwood, J.D Glazier, M.K. Sides and M. Turner, Department of Child Health and School of Biological Sciences, University of Manchester, St Mary’s Hospital, Manchester, Ml3 OJH, UK
Obiective ; Recent reports have suggested that many proliferating normal cells express telomerase activity, although telomerase activity was considered to be specific for cancer cells. These results indicate that telomerase activity is not restricted to cancer cells and that the somatic expression of telomerase is likely to be widespread. It is therefore interesting to investigate telomerase activity in trophoblasts which have characteristics of proliferation, differentiation and invasion. Methods ; Subpopulations of trophoblasts were separated by a modification of Kbman~s method and MACS (magnetic cell sorting) with antibodies of integrins. Telomerase activity was assayed semiquantitatively using fluorescence-based TRAP (modified telometic repeat amplification protocol). A cytological detection of telomerase activity was also examined by newly developed in situ TRAP procedure and the expression of human telomerase reverse transcriptase subunit (hTRT) was detected by RT-PCR. Results; Telomerase activity was detected in 43% of normal placental tissues with a tendency for its activity to decrease in accordance with gestational age. These telomerase activities showed a close correlation with expression of hTRT. In subpopulation of cytotrophoblasts (CTs) separated from early and late placenta, telomerase activity was predominantly detected in column CTs from early placenta. Fluorescence signals after in situ TRAP were observed in the trophoblastic cell layers in early placenta as well as those separated CTs, but not in late placenta. Conclusion ; These results suggest that telomerase activity correlates with the regulation of trophoblastic cell proliferation. We confirmed that in situ TRAP was useful for the detection of telometase positive cells.
By analogy with other tissues it is likely that there is acute regulation of ion transport across the placenta in response to e.g. changes in maternal and fetal plasma volume and in intracellular and extracellular osmotic pressure. Indeed, we have previously reported rapid effects of adrenaline on ion transport across the pig placenta in ~$0 and in vitro. Here we address the question of whether Cl- and K transport by the human placental syncytiotrophoblast can be acutely regulated. As regards C1’, regulatahle transport proteins are present in cytotrophoblast cells and syncytiotrop$blast; whole cell patch clamp studies show the presence of Ca -activated and volumeactivated Cl- currents; the cystic fibrosis transmembrane conductance regulator, a CAMP activated CI’ channel is expressed by the placenta and hCG can activate C1’ currents in the cytotrophoblast cell. Direct evidence of regulation comes from studies in which we fmd that increasing cell volume (induced by lowering extracellular osmolality) and raising [Ca”], (addition of the ionophore A23 187) both increase “CI’ efffux from cytotrophoblast cells pre-loaded with the tracer; interestingly addition of a CAMP ‘cocktail’ had no effect on efflux. As regards K’, we also have pvidence that an increase in cell volume and a rise in [Ca*‘li increase 6Rb (used as a marker of K+) eflux from cytotrophoblast cells and frpents of term placental villi. A variety of hormones increase [Ca Ii of the syncytiotrophoblast, including angiotensin II (AH), ATP and acetylcholine. We have recently found that AIIgnd ATP, but not acetylcholine, cause a dose dependent increase in Rb efflux from vilhts fragments. AI1 induced a maximum increase in effhtx of 18+4% (mean* s.e.; n=S) at 1OnM and caused a peak increase of48*15% at lOurn. ATP caused a peak increase in efflux of 38*8% at IOOuM. These data suggest that there is acute hormonal regulation of C1’ and K’ transport by the human placenta. Such effects are likely to be important in fetal homeostasis. Supported by the Medical Research Council.
NITRIC OXIDE AND OTHER REACTIVE OXYGEN SPECIES REGULATE HUMAN-FETAL PLACENTAL VASCUIAR RESISTANCE. L Mvatt, W. Kosaenjans, A Eis, D. Brockman. Department of Obstetrics and Gynscotogy, Unlvershy of Cincinnati College of Medicine, Cincinnati, OH 45267 USA
THE REGULATION OF MATERNAL PLACENTAL CIRCULATION. A.M. Carter, Department of Physiology, University of Odense, Winslowparken 19, Odense, Denmark.
The nitric oxide radical (NO) maintains low vascular resistance and attenuates the action of vasoconstrictors In the human fetal-placental vascular. The superoxide radical (0;) inactivatea NO but if both 0; and NO are produced in sxcess, they interact to form the long-lived powerful oxidant peroxynftritq ONOO; which can cause vascular dysfunction. Increased formation and action of ON00 vtsuaiized as nitrotyrosine residues is seen In the fetal-placental vasculature of pregnancies complicated by nitrotyrossne preeclampsla, Intrauterine growthrestriction (IUGR) andinsullndspendentdlabetesmayindlcate vascular damage leading toincreased resistance toflow Sean in these pregnancies. To assess the functionality of peroxynitrite action In the fetal-placental vasculature, we measured responses to the vasodilators prostacyctin (PGI,) and the NO donor nttroglycerinq or the vasoconstrictors angiotensin II and the thromboxane mimetic U49519 (all lo-+-19%) in isolated perfused placental cotyledons (n=5 preparations for each treatment) before or following perfuslon with authentic ON00 (10%) for 30 minutes. Following treatment with ONOO’, vasodilator responses to PGI, and nitroprusslde were significantly attenuated (p<0925ANOVA) as was thevascconstrictor response to U45919 (p~O.991 ANOVA). However, the vasoconstrictor response to All was unaffected by ON00 treatment. Attenuation of vasodilator responses to PGI, and the NO donor was greater than the vasoconstrlctor response to U49919, mimicking the altered vasoactive responses reported in preeclampsla These findings suggest that the oxtdatlve stress of preeclampsia, IUGR and diabetic pregnancies leads to production of the powerful oxidant peroxynitrite which can cause endothelial dysfunction/vascular damage leading to altered vascular reactivity and blood flows In the placenta
The human placenta is hemochorial; consequently, it lacks the arterioles that elsewhere regulate vascular resistance and blood flow. Regulation of uteroplacental blood flow, if it occurs, must be at the level of the intramyometrial arteries. Myometrial blood vessels do receive innervation, but the EC,, for noradrenaline is in the uM range and sympathetic vasoconstriction likely does not play a key role. The most potent vasoconstrictors are two hormones, arginine vasopressin and angiotensin II. The intramyometrial arteries are exquisitely sensitive to AVP, with an EC,, of 60-100 pM. However, the concentration of free AVP in plasma is even lower (l-2 PM), being reduced in the course of pregnancy. Plasma concentrations of angiotensin II in normotensive women are in the pM range compared to an EC,, in the nM range, so the renin-angiotensin system is unlikely to participate in the physiological control of uteroplacental blood flow. Intramyometrial arteries are more sensitive to thromboxane and endothelin-I than to noradrenaline, less so than to AVP. Endothelin-1 is an effective constrictor of intramyometrial arteries at nM concentrations. Substance P and vasoactive intestinal peptide are neurotransmitters in the genital tract; both are able to dilate intramyometrial arteries, and substance P is effective at pM doses. There is an endothelium-derived relaxing factor in uterine arteries. In addition, nitric oxide synthase is present in mural trophoblast.
(1998),
Vol. 19