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Heme Oxygenase Activity in Term Human Placenta
Placenta (2000), 21, 870–873 doi:10.1053/plac.2000.0574, available online at http://www.idealibrary.com on
SHORT COMMUNICATION Heme Oxygenase Activity in Term Human Placenta B. E. McLaughlina, J. M. Hutchinsona, C. H. Grahamb, G. N. Smitha,b,c, G. S. Marksa, K. Nakatsua and J. F. Briena,d Departments of a Pharmacology and Toxicology, b Anatomy and Cell Biology and c Obstetrics and Gynaecology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada Paper accepted 5 May 2000
Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P<0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme–CO (HO) pathway plays a complementary role with the -arginine–nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics. 2000 Harcourt Publishers Ltd Placenta (2000), 21, 870–873
INTRODUCTION It has been proposed that endogenous carbon monoxide (CO), produced during the heme oxygenase (HO)-catalysed oxidation of heme, plays a key role in cell function in many organ systems, including vasodilator action in the cardiovascular system (Marks et al., 1991). In the placenta, fetoplacental and uteroplacental blood flow can be regulated by formation of -arginine-derived nitric oxide (NO) (Myatt et al., 1992; Hull, White and Pearce, 1994; Ramsay et al., 1994) catalysed primarily by the nitric oxide synthase (NOS) type III isoform (Myatt et al., 1993; Conrad et al., 1993). Heme-derived CO may also play a role in the regulation of fetoplacental and uteroplacental haemodynamics. Recent investigations on the HO system have demonstrated HO enzymatic activity in guinea pig placenta (Odrcich et al., 1998) and the expression of HO protein in guinea-pig and human placenta (Odrcich et al., 1998; Lyall et al., 2000). Furthermore, in an in vitro study of the intact human placenta, selective inhibition of the HO enzymatic system increases perfusion pressure (Lyall et al., 2000). Also, there is HO d
To whom correspondence should be addressed at: Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Tel: +1(613) 533-6114; Fax: +1(613) 533-6412; E-mail: [email protected] 0143–4004/00/080870+04 $35.00/0
enzymatic activity in the human umbilical cord, including the umbilical artery and vein (Vreman et al., 2000). In order to elucidate further the physiological role of HO in the human placenta, the objective of this study was to determine HO enzymatic activity in vitro in different regions of term placenta. A gas-chromatographic method was used to measure CO formation during HO-catalysed oxidation of heme (Vreman and Stevenson, 1988).
MATERIALS AND METHODS Haemin, ethanolamine, bovine serum albumin (BSA) and NADPH were obtained from Sigma Chemical Co. (St Louis, MO, USA). Chromium mesoporphyrin (CrMP) was purchased from Porphyrin Products Inc. (Logan, UT, USA). All other chemicals were obtained from BDH Inc. (Toronto, ON, Canada) and were at least reagent-grade. Working solutions of methemalbumin and CrMP were prepared as described previously (Odrcich et al., 1998). Human placentae were obtained from elective caesareansection delivery of normal term pregnancies. Within 1 h of delivery, the following regions of the placenta were dissected: chorionic plate, chorionic villi, basal plate, amnion and choriodecidua. These placental regions were selected because they 2000 Harcourt Publishers Ltd
McLaughlin et al.: Heme Oxygenase Activity in Human Placenta
120 100 80 60 c
20 0
b NADPH
–NADPH
Figure 1. CO formation in the microsomal fraction of chorionic villi homogenate of term human placenta. CO formation was determined in the presence of 500 NADPH (NADPH), the absence of NADPH (-NADPH), or the presence of 50 CrMP plus 500 NADPH (CrMP, NADPH). The data are presented as group meanss.d. for five placentae. Group means with different letters are statistically different from each other.
represent important fetal or maternal–fetal tissues of the placenta, and are readily dissected. The microsomal fraction of 15 per cent (w/v) homogenate of each placental region was prepared, and protein content was determined as described previously (Odrcich et al., 1998). The microsomal preparation was stored at 70C for up to four weeks. HO enzymatic activity was determined in vitro by measuring the rate of CO formation during the NADPH-dependent oxidative biotransformation of heme (Vreman and Stevenson, 1988). The experimental conditions for the measurement of HO activity were optimized for the human placenta, and were similar to those conditions used for the guinea-pig placenta (Odrcich et al., 1998). In brief, for each placental region, a 1.0-ml reaction mixture containing phosphate buffer (100 m), microsomal protein (0.2 mg/ml) and methemalbumin (25 haemin and 2.5 BSA) was pipetted into a 3.5-ml amber vial. Each vial was sealed and then pre-incubated for 5 min in the dark at 37C in a shaking water bath. NADPH (500 ) was added to each sample vial; the headspace gas was displaced with CO-free air; and the incubation was continued for another 15 min. The vial was placed on powdered dry ice to stop the reaction. HO activity was measured in triplicate for each placental region. For a sample blank, a fourth vial was prepared for each placental region, in which NADPH was not added to the reaction mixture. The effect of CrMP (50 ), a selective, competitive inhibitor of HO (Vreman, Ekstrand and Stevenson, 1993; Appleton et al., 1999), on heme-derived CO formation was determined for each placental region. CO in the headspace gas was quantitated by gas–solid chromatography as described previously (Vreman and Stevenson, 1988; Odrcich et al., 1998). The amount of CO in the headspace gas was determined by interpolating the peak area value of the CO chromatographic signal on the linear CO standard curve (11–170 pmol CO). NADPH-dependent formation of CO was calculated by subtracting the value for CO produced in the placental sample
2.5
a
A
a a
2.0
a
1.5 b 1.0 0.5 0
CrMP, NADPH
10.0 Microsomal protein content (mg protein/g tissue)
40
HO activity (nmol CO/mg protein/h)
a
CP
CV
BP
AM
C–D c
B a, c
8.0
a, c
a 6.0 b
4.0 2.0 0 25
HO activity (nmol CO/g tissue/h)
CO formation (pmol CO/15 min)
140
871
CP C
CV
AM
C–D
a a
20 15
BP
a a
10 b 5 0
CP
CV
BP
AM
C–D
Figure 2. HO enzymatic activity in the microsomal fraction of homogenate of chorionic plate (CP), chorionic villi (CV), basal plate (BP), amnion (AM) and chorio-decidua (C-D) of term human placenta. (A) HO activity expressed relative to microsomal protein; (B) Microsomal protein content; (C) HO activity expressed relative to tissue wet weight. The data are presented as group meanss.d. for five placentae. Group means with different letters are statistically different from each other.
not containing NADPH (blank) from the value for CO formed in the tissue sample containing NADPH. HO enzymatic activity in the microsomal fraction of homogenate of each placental region was expressed as nmol CO formed/mg microsomal protein/h or nmol CO formed/g tissue wet weight/h. The HO enzymatic activity data for the five placental regions are presented as group meanss.d. for five human placentae. Parametric statistical analysis of the data was conducted by randomized-design, one-way ANOVA, after confirming
872
homogeneity of variance of the data by Bartlett’s test. For a statistically significant F-statistic (P<0.05), a post hoc Newman–Keuls test was conducted to determine which experimental groups were different.
RESULTS AND DISCUSSION CO formation in the microsomal fraction of homogenate of chorionic villi was NADPH-dependent and was primarily due to HO enzymatic activity, as demonstrated by 786 per cent inhibition of heme-derived CO formation by CrMP, a competitive inhibitor of HO (Vreman, Ekstrand and Stevenson, 1993; Appleton et al., 1999) (Figure 1). A higher CrMP concentration would appear to be required to produce further competitive inhibition of exogenous heme-derived CO formation. Similar results were found for the chorionic plate, basal plate, amnion and chorio-decidua (data not presented). HO activity, expressed relative to microsomal protein, was found to be similar in the chorionic plate, chorionic villi, basal plate and amnion, but not the chorio-decidua, in which enzymatic activity was lower (P<0.05) [Figure 2(A)]. These HO activity data for term human placenta, obtained by elective caesarean section, are similar to enzymatic activity data for the guinea-pig placenta at gestational day 62 (term, approximately 68 days) (Odrcich et al., 1998). As concentration of protein can vary between different tissues and may not be related to tissue concentration of HO enzyme, HO activity also was expressed relative to tissue wet weight as described previously (Vreman et al., 2000). This method of expressing HO activity provides an index of the concentration of enzymatic activity in a particular tissue, and appears to be more appropriate when comparing HO activity
Placenta (2000), Vol. 21
among different tissues. In the present study, it was found that the microsomal protein content, measured as milligram microsomal protein per gram tissue wet weight, was lower (P<0.05) in the amnion compared with the other four placental regions studied [Figure 2(B)]. HO activity, expressed relative to tissue wet weight, was higher (P<0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion [Figure 2(C)]. HO activity of the chorionic villi, a highly vascularized region of the placenta, is five-fold higher than HO activity of human umbilical artery and vein (Vreman et al., 2000), when enzymatic activity is expressed relative to tissue wet weight. The finding of HO activity in different regions of term human placenta indicates that the heme–CO (HO) pathway plays a key role in the regulation of placental blood perfusion. In summary, there was measurable HO enzymatic activity in the chorionic plate, chorionic villi, basal plate, amnion and chorio-decidua of term human placenta. Recent immunohistochemical studies of human placenta have demonstrated the presence of HO-1, the inducible isoform (Maines, 1992), in the adventitia of stem chorionic villi (Odrcich et al., 1998) and cytotrophoblast cells in the placental bed (Lyall et al., 2000). The presence of HO-2, the non-inducible isoform (Maines, 1992), has been demonstrated in the syncytiotrophoblast layer and endothelial cells of the placenta, and cytotrophoblast cells in the placental bed (Lyall et al., 2000). Taken together, the in vitro enzymatic activity and immunohistochemical localization data suggest that the heme–CO (HO) pathway may play a complementary role with the -arginine–NO (NOS) pathway in the regulation of placental haemodynamics. To evaluate further the physiological role with the HO pathway, it will be important to determine the capacity of the intact placenta for heme-derived CO formation under normal and pathophysiological (e.g. hypoxia) conditions.
ACKNOWLEDGEMENTS This research was supported by operating grants from the Heart and Stroke Foundation of Ontario (grant numbers T-3361 and NA-4438). Dr Charles Graham is the recipient of a Heart and Stroke Foundation of Canada Research Scholarship. The authors wish to thank Drs Hendrik J. Vreman and David K. Stevenson, Department of Pediatrics, Stanford University School of Medicine, USA, for the use of their gas chromatograph to quantitate CO formation and for their continuing interest in our research. The authors gratefully acknowledge the insight of Dr Vreman in the analysis and presentation of the HO enzymatic activity findings.
REFERENCES Appleton SD, Chretien ML, McLaughlin BE, Vreman HJ, Stevenson DK, Brien JF, Nakatsu K, Maurice DH & Marks GS (1999) Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations. Drug Metab Dispos, 27, 1214–1219. Conrad KP, Vill M, McGuire PG, Dail WG & Davis AK (1993) Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi. FASEB J, 7, 1269–1276. Hull AD, White CR & Pearce WJ (1994) Endothelium-derived relaxing factor and cyclic GMP-dependent vasorelaxation in human chorionic plate arteries. Placenta, 15, 365–375. Lyall F, Barber A, Myatt L, Bulmer JN & Robson SC (2000) Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function. FASEB J, 14, 208–219.
Maines MD (1992) Heme Oxygenase: Clinical Applications and Functions. Boca Raton: CRC Press. 276 pp. Marks GS, Brien JF, Nakatsu K & McLaughlin BE (1991) Does carbon monoxide have a physiological function? Trends Pharmacol Sci, 12, 185–188. Myatt L, Brewer AS, Langdon G & Brockman DE (1992) Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol, 166, 224–230. Myatt L, Brockman DE, Langdon G & Pollock JS (1993) Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree. Placenta, 14, 373–383. Odrcich MJ, Graham CH, Kimura KA, McLaughlin BE, Marks GS, Nakatsu K & Brien JF (1998) Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation. Placenta, 19, 509–516.
McLaughlin et al.: Heme Oxygenase Activity in Human Placenta Ramsay B, de Belder A, Campbell S, Moncada S & Martin JF (1994) A nitric oxide donor improves uterine artery diastolic blood flow in normal early pregnancy and in women at high risk of pre-eclampsia. Eur J Clin Invest, 24, 76–78. Vreman HJ & Stevenson DK (1988) Heme oxygenase activity as measured by carbon monoxide production. Analyt Biochem, 168, 31–38.
873 Vreman HJ, Ekstrand BC & Stevenson DK (1993) Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatric Res, 33, 195–200. Vreman HJ, Wong RJ, Kim EC, Nabseth, DC, Marks GS & Stevenson DK (2000) Heme oxygenase activity in human umbilical cord and rat vascular tissues. Placenta, 21, 337–344.
SHORT COMMUNICATION Heme Oxygenase Activity in Term Human Placenta B. E. McLaughlina, J. M. Hutchinsona, C. H. Grahamb, G. N. Smitha,b,c, G. S. Marksa, K. Nakatsua and J. F. Briena,d Departments of a Pharmacology and Toxicology, b Anatomy and Cell Biology and c Obstetrics and Gynaecology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada Paper accepted 5 May 2000
Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P<0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme–CO (HO) pathway plays a complementary role with the -arginine–nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics. 2000 Harcourt Publishers Ltd Placenta (2000), 21, 870–873
INTRODUCTION It has been proposed that endogenous carbon monoxide (CO), produced during the heme oxygenase (HO)-catalysed oxidation of heme, plays a key role in cell function in many organ systems, including vasodilator action in the cardiovascular system (Marks et al., 1991). In the placenta, fetoplacental and uteroplacental blood flow can be regulated by formation of -arginine-derived nitric oxide (NO) (Myatt et al., 1992; Hull, White and Pearce, 1994; Ramsay et al., 1994) catalysed primarily by the nitric oxide synthase (NOS) type III isoform (Myatt et al., 1993; Conrad et al., 1993). Heme-derived CO may also play a role in the regulation of fetoplacental and uteroplacental haemodynamics. Recent investigations on the HO system have demonstrated HO enzymatic activity in guinea pig placenta (Odrcich et al., 1998) and the expression of HO protein in guinea-pig and human placenta (Odrcich et al., 1998; Lyall et al., 2000). Furthermore, in an in vitro study of the intact human placenta, selective inhibition of the HO enzymatic system increases perfusion pressure (Lyall et al., 2000). Also, there is HO d
To whom correspondence should be addressed at: Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Tel: +1(613) 533-6114; Fax: +1(613) 533-6412; E-mail: [email protected] 0143–4004/00/080870+04 $35.00/0
enzymatic activity in the human umbilical cord, including the umbilical artery and vein (Vreman et al., 2000). In order to elucidate further the physiological role of HO in the human placenta, the objective of this study was to determine HO enzymatic activity in vitro in different regions of term placenta. A gas-chromatographic method was used to measure CO formation during HO-catalysed oxidation of heme (Vreman and Stevenson, 1988).
MATERIALS AND METHODS Haemin, ethanolamine, bovine serum albumin (BSA) and NADPH were obtained from Sigma Chemical Co. (St Louis, MO, USA). Chromium mesoporphyrin (CrMP) was purchased from Porphyrin Products Inc. (Logan, UT, USA). All other chemicals were obtained from BDH Inc. (Toronto, ON, Canada) and were at least reagent-grade. Working solutions of methemalbumin and CrMP were prepared as described previously (Odrcich et al., 1998). Human placentae were obtained from elective caesareansection delivery of normal term pregnancies. Within 1 h of delivery, the following regions of the placenta were dissected: chorionic plate, chorionic villi, basal plate, amnion and choriodecidua. These placental regions were selected because they 2000 Harcourt Publishers Ltd
McLaughlin et al.: Heme Oxygenase Activity in Human Placenta
120 100 80 60 c
20 0
b NADPH
–NADPH
Figure 1. CO formation in the microsomal fraction of chorionic villi homogenate of term human placenta. CO formation was determined in the presence of 500 NADPH (NADPH), the absence of NADPH (-NADPH), or the presence of 50 CrMP plus 500 NADPH (CrMP, NADPH). The data are presented as group meanss.d. for five placentae. Group means with different letters are statistically different from each other.
represent important fetal or maternal–fetal tissues of the placenta, and are readily dissected. The microsomal fraction of 15 per cent (w/v) homogenate of each placental region was prepared, and protein content was determined as described previously (Odrcich et al., 1998). The microsomal preparation was stored at 70C for up to four weeks. HO enzymatic activity was determined in vitro by measuring the rate of CO formation during the NADPH-dependent oxidative biotransformation of heme (Vreman and Stevenson, 1988). The experimental conditions for the measurement of HO activity were optimized for the human placenta, and were similar to those conditions used for the guinea-pig placenta (Odrcich et al., 1998). In brief, for each placental region, a 1.0-ml reaction mixture containing phosphate buffer (100 m), microsomal protein (0.2 mg/ml) and methemalbumin (25 haemin and 2.5 BSA) was pipetted into a 3.5-ml amber vial. Each vial was sealed and then pre-incubated for 5 min in the dark at 37C in a shaking water bath. NADPH (500 ) was added to each sample vial; the headspace gas was displaced with CO-free air; and the incubation was continued for another 15 min. The vial was placed on powdered dry ice to stop the reaction. HO activity was measured in triplicate for each placental region. For a sample blank, a fourth vial was prepared for each placental region, in which NADPH was not added to the reaction mixture. The effect of CrMP (50 ), a selective, competitive inhibitor of HO (Vreman, Ekstrand and Stevenson, 1993; Appleton et al., 1999), on heme-derived CO formation was determined for each placental region. CO in the headspace gas was quantitated by gas–solid chromatography as described previously (Vreman and Stevenson, 1988; Odrcich et al., 1998). The amount of CO in the headspace gas was determined by interpolating the peak area value of the CO chromatographic signal on the linear CO standard curve (11–170 pmol CO). NADPH-dependent formation of CO was calculated by subtracting the value for CO produced in the placental sample
2.5
a
A
a a
2.0
a
1.5 b 1.0 0.5 0
CrMP, NADPH
10.0 Microsomal protein content (mg protein/g tissue)
40
HO activity (nmol CO/mg protein/h)
a
CP
CV
BP
AM
C–D c
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a, c
a 6.0 b
4.0 2.0 0 25
HO activity (nmol CO/g tissue/h)
CO formation (pmol CO/15 min)
140
871
CP C
CV
AM
C–D
a a
20 15
BP
a a
10 b 5 0
CP
CV
BP
AM
C–D
Figure 2. HO enzymatic activity in the microsomal fraction of homogenate of chorionic plate (CP), chorionic villi (CV), basal plate (BP), amnion (AM) and chorio-decidua (C-D) of term human placenta. (A) HO activity expressed relative to microsomal protein; (B) Microsomal protein content; (C) HO activity expressed relative to tissue wet weight. The data are presented as group meanss.d. for five placentae. Group means with different letters are statistically different from each other.
not containing NADPH (blank) from the value for CO formed in the tissue sample containing NADPH. HO enzymatic activity in the microsomal fraction of homogenate of each placental region was expressed as nmol CO formed/mg microsomal protein/h or nmol CO formed/g tissue wet weight/h. The HO enzymatic activity data for the five placental regions are presented as group meanss.d. for five human placentae. Parametric statistical analysis of the data was conducted by randomized-design, one-way ANOVA, after confirming
872
homogeneity of variance of the data by Bartlett’s test. For a statistically significant F-statistic (P<0.05), a post hoc Newman–Keuls test was conducted to determine which experimental groups were different.
RESULTS AND DISCUSSION CO formation in the microsomal fraction of homogenate of chorionic villi was NADPH-dependent and was primarily due to HO enzymatic activity, as demonstrated by 786 per cent inhibition of heme-derived CO formation by CrMP, a competitive inhibitor of HO (Vreman, Ekstrand and Stevenson, 1993; Appleton et al., 1999) (Figure 1). A higher CrMP concentration would appear to be required to produce further competitive inhibition of exogenous heme-derived CO formation. Similar results were found for the chorionic plate, basal plate, amnion and chorio-decidua (data not presented). HO activity, expressed relative to microsomal protein, was found to be similar in the chorionic plate, chorionic villi, basal plate and amnion, but not the chorio-decidua, in which enzymatic activity was lower (P<0.05) [Figure 2(A)]. These HO activity data for term human placenta, obtained by elective caesarean section, are similar to enzymatic activity data for the guinea-pig placenta at gestational day 62 (term, approximately 68 days) (Odrcich et al., 1998). As concentration of protein can vary between different tissues and may not be related to tissue concentration of HO enzyme, HO activity also was expressed relative to tissue wet weight as described previously (Vreman et al., 2000). This method of expressing HO activity provides an index of the concentration of enzymatic activity in a particular tissue, and appears to be more appropriate when comparing HO activity
Placenta (2000), Vol. 21
among different tissues. In the present study, it was found that the microsomal protein content, measured as milligram microsomal protein per gram tissue wet weight, was lower (P<0.05) in the amnion compared with the other four placental regions studied [Figure 2(B)]. HO activity, expressed relative to tissue wet weight, was higher (P<0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion [Figure 2(C)]. HO activity of the chorionic villi, a highly vascularized region of the placenta, is five-fold higher than HO activity of human umbilical artery and vein (Vreman et al., 2000), when enzymatic activity is expressed relative to tissue wet weight. The finding of HO activity in different regions of term human placenta indicates that the heme–CO (HO) pathway plays a key role in the regulation of placental blood perfusion. In summary, there was measurable HO enzymatic activity in the chorionic plate, chorionic villi, basal plate, amnion and chorio-decidua of term human placenta. Recent immunohistochemical studies of human placenta have demonstrated the presence of HO-1, the inducible isoform (Maines, 1992), in the adventitia of stem chorionic villi (Odrcich et al., 1998) and cytotrophoblast cells in the placental bed (Lyall et al., 2000). The presence of HO-2, the non-inducible isoform (Maines, 1992), has been demonstrated in the syncytiotrophoblast layer and endothelial cells of the placenta, and cytotrophoblast cells in the placental bed (Lyall et al., 2000). Taken together, the in vitro enzymatic activity and immunohistochemical localization data suggest that the heme–CO (HO) pathway may play a complementary role with the -arginine–NO (NOS) pathway in the regulation of placental haemodynamics. To evaluate further the physiological role with the HO pathway, it will be important to determine the capacity of the intact placenta for heme-derived CO formation under normal and pathophysiological (e.g. hypoxia) conditions.
ACKNOWLEDGEMENTS This research was supported by operating grants from the Heart and Stroke Foundation of Ontario (grant numbers T-3361 and NA-4438). Dr Charles Graham is the recipient of a Heart and Stroke Foundation of Canada Research Scholarship. The authors wish to thank Drs Hendrik J. Vreman and David K. Stevenson, Department of Pediatrics, Stanford University School of Medicine, USA, for the use of their gas chromatograph to quantitate CO formation and for their continuing interest in our research. The authors gratefully acknowledge the insight of Dr Vreman in the analysis and presentation of the HO enzymatic activity findings.
REFERENCES Appleton SD, Chretien ML, McLaughlin BE, Vreman HJ, Stevenson DK, Brien JF, Nakatsu K, Maurice DH & Marks GS (1999) Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations. Drug Metab Dispos, 27, 1214–1219. Conrad KP, Vill M, McGuire PG, Dail WG & Davis AK (1993) Expression of nitric oxide synthase by syncytiotrophoblast in human placental villi. FASEB J, 7, 1269–1276. Hull AD, White CR & Pearce WJ (1994) Endothelium-derived relaxing factor and cyclic GMP-dependent vasorelaxation in human chorionic plate arteries. Placenta, 15, 365–375. Lyall F, Barber A, Myatt L, Bulmer JN & Robson SC (2000) Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function. FASEB J, 14, 208–219.
Maines MD (1992) Heme Oxygenase: Clinical Applications and Functions. Boca Raton: CRC Press. 276 pp. Marks GS, Brien JF, Nakatsu K & McLaughlin BE (1991) Does carbon monoxide have a physiological function? Trends Pharmacol Sci, 12, 185–188. Myatt L, Brewer AS, Langdon G & Brockman DE (1992) Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol, 166, 224–230. Myatt L, Brockman DE, Langdon G & Pollock JS (1993) Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree. Placenta, 14, 373–383. Odrcich MJ, Graham CH, Kimura KA, McLaughlin BE, Marks GS, Nakatsu K & Brien JF (1998) Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation. Placenta, 19, 509–516.
McLaughlin et al.: Heme Oxygenase Activity in Human Placenta Ramsay B, de Belder A, Campbell S, Moncada S & Martin JF (1994) A nitric oxide donor improves uterine artery diastolic blood flow in normal early pregnancy and in women at high risk of pre-eclampsia. Eur J Clin Invest, 24, 76–78. Vreman HJ & Stevenson DK (1988) Heme oxygenase activity as measured by carbon monoxide production. Analyt Biochem, 168, 31–38.
873 Vreman HJ, Ekstrand BC & Stevenson DK (1993) Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatric Res, 33, 195–200. Vreman HJ, Wong RJ, Kim EC, Nabseth, DC, Marks GS & Stevenson DK (2000) Heme oxygenase activity in human umbilical cord and rat vascular tissues. Placenta, 21, 337–344.