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V.I.P. receptors, positively coupled with adenylate cyclase activity on fetal vascularization of human placenta
.4bstral ts: European Placenta Group
471
pH 7.3-7.4) was passed under open circuit, and ion selective electrodes used to measure inflow and outflow concentrations. After an initial perfusion period perfusate containing inhibitor was passed for 15 min. Dinitrophenol (IO-~ M) caused a significant release of K+ and increase in Na+ and Ca.Z+ uptake by tissue on both circuits. Ouabain (IO- M) caused a significant release of K + on both circuits and had no detectable effect on Ca’ + or maternal Na+. A small but significant release of Na+ occurred on the fetal circuit. Ca*+ movements showed a dependence on perfusate CaZ + concentration similar to that observed in previous closed circuit studies. These results support the view that CaZ+ crosses the placenta from mother to fetus by a membrane bound ATPase system and that a CaZ+/Na+ exchanger is not involved with this process. (Supported by Action Research.)
FETAL PLACENTAL BLOOD FLOW AFTER UTERINE ARTERY LIGATION IN THE GUINEA-PIG A. M. Carter & A. Detmer (Department of Physiology and Biomedical Laboratory, University of Odense, Campusvej 55, DK-5230 Odense, Denmark) Ligation of the uterine artery in the pregnant guinea-pig causes a reduction in placental mass and maternal placental glood flow and leads to retardation of fetal growth (IUGR). We asked how fetal placental blood flow behaved in this situation, since it is an important determinant of oxygen and substrate delivery to the fetus. The uterine artery to one horn was ligated on day 30-33 of gestation. On day 60-64 we studied a fetus with IUGR and asymmetric growth (brain: liver weight ratio 0.9) or a control fetus from the contralateral horn. To measure placental blood flow, tracer microspheres were injected into the right atrium of the fetus and a reference sample of blood was withdrawn from the vitelline artery. IUGR fetuses (n = 5) weighed 56.6 f 2.9 g (mean f se.) and the controls (n =6) weighed 83.0 f 3.5 g (p = 0.001). The corresponding placental weights were 3.2 f 0.2 and 5.2 f 0.4g (p = 0.001). Heart rate was lower in IUGR fetuses than in controls, 206 f 19 against 281 f 7 beats/min (p = 0.01). Haemoglobin concentration, measured in arterial blood, was similar in the two groups. Fetal placental blood flow was 4.9 f 1.2 ml/min in IUGR fetuses compared to 9.9 f 1.5 ml/min in controls (p = 0.05). There was no significant difference between the placental perfusion rates, which were I .5 f 0.2 and 1.9 f 0.3 ml/min per g tissue. It is known that uterine artery ligation restricts placental growth before affecting fetal weight (Jansson et al, Biologiu Neonatorum, 49, 172-180). Our results show that the decrease in placental size is accompanied by a reduction in fetal placental blood flow. It is therefore suggested that the consequent limitation upon oxygen delivery to the fetus is a causative factor in growth retardation. This interpretation is supported by experiments on the fetal lamb, where the long-term effects of a reduction in umbilical blood flow and oxygen delivery are a fall in fetal oxygen consumption and a decrease in the rate of fetal growth (Anderson et al, 1986, AmericanJournal OfPhysiology, 250, H1o37-H1o42).
V.I.P. RECEPTORS, POSITIVELY COUPLED WITH ADENYLATE CYCLASE ACTIVITY ON FETAL VASCULARIZATION OF HUMAN PLACENTA A. Malassine, F. Mondon”, J. Bessonb, M. ViaP, G. Tanguy, W. Rostineb 8z F. Ferrp (Laboratoire de Physiologie Cellulaire UFR Sciences Poitiers, a INSERM U. 166, Maternitt Baudelocque and bU.55 Hopital St-Antoine, Paris, France) The presence of Vasoactive Intestinal Peptide (VIP) has been reported in human placenta. a non-innervated organ. However, the exact origin of this neuropeptide, the precise localization of its binding sites and the mechanism(s) of action remain to be clarified.
Placenta (1989), Vol. IO
472
The [*251]-VIP binding to placental slices was saturable and unlabelled VIP was able to compete in a dose-dependent manner with an IC,, value of 5.2 f 1.3 10~‘~ M. On the same preparation VIP50 stimulated specifically the CAMP synthesis with an ED,, value of 2.9 f 1.6 IO-9
M.
Light microscopic autoradiography showed the association of numerous grains with the stem villi arteries and arterioles (muscle layer and endothelium). Veins, veinules and capillaries were less labelled. Coincubation of [ I2 51]-VIP with an excess of unlabelled VIP resulted in a reduction of grains. Consequently adenylate cyclase activity was studied on isolated membranes of stem villi vessels obtained by fine mechanical dissociation and ultracentrifugations. The VIP stimulated, in a dose-dependent manner, the adenylate cyclase activity of these vascular membranes. These results confirm the presence of VIP receptors positively coupled with adenylate cyclase on the fetal vascularization and suggest a functional role for the peptide in placental haemodynamics. EXTRACELLULAR ADENINE NUCLEOTIDES IN HUMAN TROPHOBLASTIC PURINE NUCLEOTIDE SYNTHESIS K. Vettenranta & K. 0. Raivio (Children’s Hospital, University of Helsinki, SF-oozgo, Finland) ATP and other purine nucleotides appear to be synthesized through the reutilization of preexisting purine bases and/or nucleosides rather than de novo synthesis in the human trophoblast throughout gestation. However, the origin of the reutilizable purines is unknown. We studied the ability of cultured human trophoblasts to use extracellular adenine nucleotides as precursors in their intracellular nucleotide synthesis. First and third trimester placentae were obtained from elective terminations and caesarean sections, respectively. Primary short-term cultures of trophoblasts were established using collagenase treatment. The cultured cells were incubated in the presence of 2 pM deoxycoformycin and [ ’ %I-ATP (I, 5 or IO PM) or [ ’ a ClADP (I, 5 or I 5 PM) for up to 4 h. Parallel experiments in the presence of 250 pM alpha-betamethylene-ADP or IO pM dipyridamole plus IO PM adenine were also performed. Purine compounds were separated using thin-layer chromatography. The metabolic integrity of the cells was monitored with the adenylate energy charge [mean 0.71 (0.5oo.89)]. Both the first and third trimester trophoblasts dephosphorylated extracellular adenine nucleotides to adenosine, which was then intracellularly rephosphorylated. Adenine nucleotides constituted more than 80 per cent of the nucleotides thus formed. The contribution of specific 5’-nucleotidase to the dephosphorylation of extracellular AMP was 7 f 6 per cent in the first and 37 f 15 per cent in the third trimester cells. Uptake of intact nucleotides did not take place. Incorporation of radioactivity into intracellular nucleotides increased as a function of time and precursor concentration in both the first and third trimester trophoblasts. The utilization of [’ 4 Cl- ADP was significantly (p < 0.05) more active in the third trimester cells while no change with gestation in that of [I’]-C-ATP was seen. In conclusion, it appears that throughout gestation the human trophoblast is able to degrade extracellular ATP and ADP to adenosine, which is further employed as a reutilizable substrate in intracellular nucleotide synthesis.
TAURINE TRANSPORT BY MICROVILLOUS MEMBRANE VESICLES AND THE PERFUSED COTYLEDON OF THE HUMAN PLACENTA P. I. Karl & S. E. Fisher (Departments of Paediatrics and Research, North Shore University Hospital-Cornell University Medical College, Manhasset, NY I 1030, USA)
471
pH 7.3-7.4) was passed under open circuit, and ion selective electrodes used to measure inflow and outflow concentrations. After an initial perfusion period perfusate containing inhibitor was passed for 15 min. Dinitrophenol (IO-~ M) caused a significant release of K+ and increase in Na+ and Ca.Z+ uptake by tissue on both circuits. Ouabain (IO- M) caused a significant release of K + on both circuits and had no detectable effect on Ca’ + or maternal Na+. A small but significant release of Na+ occurred on the fetal circuit. Ca*+ movements showed a dependence on perfusate CaZ + concentration similar to that observed in previous closed circuit studies. These results support the view that CaZ+ crosses the placenta from mother to fetus by a membrane bound ATPase system and that a CaZ+/Na+ exchanger is not involved with this process. (Supported by Action Research.)
FETAL PLACENTAL BLOOD FLOW AFTER UTERINE ARTERY LIGATION IN THE GUINEA-PIG A. M. Carter & A. Detmer (Department of Physiology and Biomedical Laboratory, University of Odense, Campusvej 55, DK-5230 Odense, Denmark) Ligation of the uterine artery in the pregnant guinea-pig causes a reduction in placental mass and maternal placental glood flow and leads to retardation of fetal growth (IUGR). We asked how fetal placental blood flow behaved in this situation, since it is an important determinant of oxygen and substrate delivery to the fetus. The uterine artery to one horn was ligated on day 30-33 of gestation. On day 60-64 we studied a fetus with IUGR and asymmetric growth (brain: liver weight ratio 0.9) or a control fetus from the contralateral horn. To measure placental blood flow, tracer microspheres were injected into the right atrium of the fetus and a reference sample of blood was withdrawn from the vitelline artery. IUGR fetuses (n = 5) weighed 56.6 f 2.9 g (mean f se.) and the controls (n =6) weighed 83.0 f 3.5 g (p = 0.001). The corresponding placental weights were 3.2 f 0.2 and 5.2 f 0.4g (p = 0.001). Heart rate was lower in IUGR fetuses than in controls, 206 f 19 against 281 f 7 beats/min (p = 0.01). Haemoglobin concentration, measured in arterial blood, was similar in the two groups. Fetal placental blood flow was 4.9 f 1.2 ml/min in IUGR fetuses compared to 9.9 f 1.5 ml/min in controls (p = 0.05). There was no significant difference between the placental perfusion rates, which were I .5 f 0.2 and 1.9 f 0.3 ml/min per g tissue. It is known that uterine artery ligation restricts placental growth before affecting fetal weight (Jansson et al, Biologiu Neonatorum, 49, 172-180). Our results show that the decrease in placental size is accompanied by a reduction in fetal placental blood flow. It is therefore suggested that the consequent limitation upon oxygen delivery to the fetus is a causative factor in growth retardation. This interpretation is supported by experiments on the fetal lamb, where the long-term effects of a reduction in umbilical blood flow and oxygen delivery are a fall in fetal oxygen consumption and a decrease in the rate of fetal growth (Anderson et al, 1986, AmericanJournal OfPhysiology, 250, H1o37-H1o42).
V.I.P. RECEPTORS, POSITIVELY COUPLED WITH ADENYLATE CYCLASE ACTIVITY ON FETAL VASCULARIZATION OF HUMAN PLACENTA A. Malassine, F. Mondon”, J. Bessonb, M. ViaP, G. Tanguy, W. Rostineb 8z F. Ferrp (Laboratoire de Physiologie Cellulaire UFR Sciences Poitiers, a INSERM U. 166, Maternitt Baudelocque and bU.55 Hopital St-Antoine, Paris, France) The presence of Vasoactive Intestinal Peptide (VIP) has been reported in human placenta. a non-innervated organ. However, the exact origin of this neuropeptide, the precise localization of its binding sites and the mechanism(s) of action remain to be clarified.
Placenta (1989), Vol. IO
472
The [*251]-VIP binding to placental slices was saturable and unlabelled VIP was able to compete in a dose-dependent manner with an IC,, value of 5.2 f 1.3 10~‘~ M. On the same preparation VIP50 stimulated specifically the CAMP synthesis with an ED,, value of 2.9 f 1.6 IO-9
M.
Light microscopic autoradiography showed the association of numerous grains with the stem villi arteries and arterioles (muscle layer and endothelium). Veins, veinules and capillaries were less labelled. Coincubation of [ I2 51]-VIP with an excess of unlabelled VIP resulted in a reduction of grains. Consequently adenylate cyclase activity was studied on isolated membranes of stem villi vessels obtained by fine mechanical dissociation and ultracentrifugations. The VIP stimulated, in a dose-dependent manner, the adenylate cyclase activity of these vascular membranes. These results confirm the presence of VIP receptors positively coupled with adenylate cyclase on the fetal vascularization and suggest a functional role for the peptide in placental haemodynamics. EXTRACELLULAR ADENINE NUCLEOTIDES IN HUMAN TROPHOBLASTIC PURINE NUCLEOTIDE SYNTHESIS K. Vettenranta & K. 0. Raivio (Children’s Hospital, University of Helsinki, SF-oozgo, Finland) ATP and other purine nucleotides appear to be synthesized through the reutilization of preexisting purine bases and/or nucleosides rather than de novo synthesis in the human trophoblast throughout gestation. However, the origin of the reutilizable purines is unknown. We studied the ability of cultured human trophoblasts to use extracellular adenine nucleotides as precursors in their intracellular nucleotide synthesis. First and third trimester placentae were obtained from elective terminations and caesarean sections, respectively. Primary short-term cultures of trophoblasts were established using collagenase treatment. The cultured cells were incubated in the presence of 2 pM deoxycoformycin and [ ’ %I-ATP (I, 5 or IO PM) or [ ’ a ClADP (I, 5 or I 5 PM) for up to 4 h. Parallel experiments in the presence of 250 pM alpha-betamethylene-ADP or IO pM dipyridamole plus IO PM adenine were also performed. Purine compounds were separated using thin-layer chromatography. The metabolic integrity of the cells was monitored with the adenylate energy charge [mean 0.71 (0.5oo.89)]. Both the first and third trimester trophoblasts dephosphorylated extracellular adenine nucleotides to adenosine, which was then intracellularly rephosphorylated. Adenine nucleotides constituted more than 80 per cent of the nucleotides thus formed. The contribution of specific 5’-nucleotidase to the dephosphorylation of extracellular AMP was 7 f 6 per cent in the first and 37 f 15 per cent in the third trimester cells. Uptake of intact nucleotides did not take place. Incorporation of radioactivity into intracellular nucleotides increased as a function of time and precursor concentration in both the first and third trimester trophoblasts. The utilization of [’ 4 Cl- ADP was significantly (p < 0.05) more active in the third trimester cells while no change with gestation in that of [I’]-C-ATP was seen. In conclusion, it appears that throughout gestation the human trophoblast is able to degrade extracellular ATP and ADP to adenosine, which is further employed as a reutilizable substrate in intracellular nucleotide synthesis.
TAURINE TRANSPORT BY MICROVILLOUS MEMBRANE VESICLES AND THE PERFUSED COTYLEDON OF THE HUMAN PLACENTA P. I. Karl & S. E. Fisher (Departments of Paediatrics and Research, North Shore University Hospital-Cornell University Medical College, Manhasset, NY I 1030, USA)