Life Sciences Vol . 23, pp . 39-44 Printed is the U .S .A .
Pergamon press
PLASMA CATECHOLAMINES IN FETAL AND NEONATAL RATS Nira Ban-Jonathan Department of Physiology Indiana University School of Medicine Tndtanapolls, Indiana 46202 (Received in final form May 12, 1978) Summary During the last day of gestation, dopamine was higher in fetal than to maternal plasma whereas norepinephrtne and epinephrine were slmtlar . Immediately after birth, plasma norepinephrine and epinephrine fell to 10x of their levels in term fetuses, remained low to the second day of life and reached adult levels within one to two weeks . Plasma dopamine, however, did not reduce much after birth . The data are consistent with the predominance of the extra-adrenal chramaffin tissue in the fetus, its postnatal involution, and the delayed maturation of the adrenal medulla in the newborn .
It has been suggested that fetal catecholamines participate to the control of placental circulation ~1), and that the most potent stimulus for their release is hypoxia (2) . Epinephrine secretion 1n the newborn animal mi ht protect it from hypoglycemic reactions that sometimes occur after birth ~3) . In view of these important cardiovascular, pulmonary and metabolic functions served by catecholamlnes, it is surprising that so little information is available on their plasma levels in fetal and newborn animals . This is probably due to the lack of sensitivity of existing methods for measuring these compounds in the minute amounts of plasma obtainable from fetal and neonatal rats . Yle have recently developed a very sensitive double isotope radioenzymatic assay which 1s capable of measuring simultaneously do amine, norepinephrine and epinephrine in as little as 25-50 ul of plasma (4~ . By utilizing this assay we demonstrated that during gestation in the rat, catecholamines were compartmentalized between the fetal and maternal clrculations and the amniotic fluid ~5) . Moreover, we found that in 18 day old fetuses, dopamine constituted as much as 30~ of total plasma catecholamtnes whereas it was no more than 4-5~ of the adult's . Thus, fit became of finterest to extend these studies and examine the changes occurring in plasma catecholamtnes immediately after birth and during the first two weeks of neonatal life .
0300-9653/78/0703-0039502 .00/0 Copynght © 1978 Pergsmon Press
40
Fatal and Neonatal Plasma Catecholaminea
Vol . 23, No . 1, 1978
Materials and Methods Male and female Wistar rats, weighing 250-300 g sere used 1n this study . Rats in proestrus were placed overnight with males and the next morning, with the appearance of vaginal sperm, was designated as day one of gestation . All animals were decapitated between 10 and 12 a .m . to eliminate diurnal variations . Trunk blood was collected into chilled heparïnized tubes and the plasma separated and stored at -20 °C . Pregnant rats were decapitated ôn day 22 of gestation which is the day of parturition . The uteri were immediately removed and the fetuses decapitated . Fetal trunk blood was collected by the aid of heparinized pasteur pipette and pooled from every 2-3 fetuses to a final volume of 200 ul . The time from decapitation of the mother to collection of blood from all the fetuses did not exceed 15 minutes . No significant differences were observed between catecholamines in plasma collected from fetuses immediately after maternal decapitation or 10-12 minutes afterwards . Newborn rats were decapitated on days 1 (the day of parturition), 2, 7 and 14 of postnatal life . Trunk blood was pooled from 1-2 neonates to a final volume of 300 ul . Catecholamines assay : Triplicate aliquots (25 or 50 ul) of plasma were incubated with catechol-0-methyl transferase and S-adenosyl-L-methionine The 0-methylated dopamine, norepi(methyl- 3 H) as described previously (4) . nephrine and epinephrine were extracted, separated by two-dimensional thin layer chrromatography, and counted in Aquasol in a liquid scinttllntion spectrometer . The assay sentitivity was 5-15 pg for each amine and the overlap between arpr two of the three compounds did not exceed 1-2x . The results were test . analyzed by a student
x
Results and Discussion As shown in Fig . 1, plasma concentrations of dopamine, norepinephrine and epinephrine 1n pregnant rats on the last day of gestation were 0 .6210 .12, These values do not 3 .7510 .59 and 9 .0511 .67 ng/ml (meanslSE), respectively . differ significantly from catecholamine levels in plasma collected from decapitated male rats . Thus, there was no apparent effect of advanced pregnancy on the levels of circulating catecholamines . On day 22 of gestation, norepinephrine and epinephrine concentrations in fetal and maternal plasma were similar . Considering that at that time, the fetal adrenal content of these compounds does not exceed 1-2X of the adult's (6), such finding raises the possibility that catecholamines transfer between the maternal and fetal circulations . However, there is a dissimilarii~y in Moreover, in our prevdopamine concentration in maternal and fetal plasma . ious study (5) we found that on days 18 and 19 of gestation, epineph M ne was 5 fold lower in fetal than in maternal plasma . Other evidence against placental transfer of catecholamines exists (7), although Parvez and Parvez (8) demonstrated an uptake of tritiated epinephrine, injected into the mother, by the fetal heart . A likely explanation for the high plasma levels of catecholamines in the fetus at term is that they primarily originate from tissues other than the adrenal . Indeed, the fetus possesses a unique structure, the extra-adrenal chromaffin tissue, which reaches its maximal structural growth at birth and On day 22 of fetal life, regresses within two weeks of postnatal life (9) . the content of norepinephrine and spinephrine in this tissue equals or exceeds that of the adrenal medulla (10) . Fetal plasma dopamine might originate either from the extra-adrenal chrbmaffin tissue or from SIF (small, intensely fluorescent) cells found in structures such as the superior cervical gan lie and the carotid bodies which contain considerable amounts of dopamine ~11) .
Fatal aad Neonatal Plasma Catecholamines
Vol . 23, No . 1, 1978
+
41
aoPSIIIRi ,_.,
,~~,
II
f-1
II
II
RORiPIRiPRRIMi
ë
~~
r
,
+_
0 ~, a i O O u 1+ r H u e a lo ~ m
RPIRiPRRIRi
(.)
PARI'Oiliplf ('I)
(6)
1
(1>)
a
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~
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FIG .
n
dv)
I"I
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a wa
II
tlq,lw (+ ao .)
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R.a.aw
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1
Plasma catacholamines at different developmental stages . Day 1 of gestation was desi nated with the presence of vaginal sperm . Day ~ of neonatal life was the day of parturition . A11 decapitations were done between 10 and 12 a .m, and trunk blood collected into heparinized tubes . Blood was pooled as follows : from 2-3 fetuses ; from 1-2 neonates and from slngle adults . Catecholamtnes were determined by a radioenzymatic assay in triplicate aliquots (25 or 50 u1) . Values are meanstSE and the number of pooled samples per group is shown in parentheses .
On days 1 and 2 of neonatal life, plasma norepinephrine and epinephrine were extra y low, compristng not more n 10~ of their levels in fet 1e plasma at term . By da~yr 7, plasma levels of these compounds increased, and by day 14 they reached or surpassed adult plasma levels . po~amine, however, dtd not fall much after parturition and was stt11 higher in plasma fr~am two week old neonates than that from mature males or pregnant finales .
42
Fetal and Neonatal Plasma Catecholaminae
Vol . 23, No . 1, 1978
Several lines of evidence indicate that glucocorticoids maintain the integrity of the extra-adrenal chromafftn tissue in fetal animals (l2, 13), as well as stimulate an increase in the activity of the norepinephrine methylating enzyme PNMT (phenylethanolamine-N-methyl transferase) both in the adrenal (14) and the extra-adrenal chromafftn tissue (15~ . The cause for the involution of the extra-adrenal chromaffin tissue immediately after birth has been attributed to the withdrawal of glucocorticoid support resulting from a reduction in their circulating levels . It has been argued that such withdrawal would not be detrimental for the maintenance of adrenal medullary cells because of their proximity to the cortex and the constant exposure to very high concentrations of glucocorticoids . Indeed, in 1966, Jost (1) demonstrated a decrease in pituitary stimulation of the adrenal cortex immediately after birth . Recently, this was supported by the finding that circulating corticosterone in the rat was reduced in the first eight hours after birth to 20~ of its levels in term fetuses (16) . Thus, it can be postulated that after parturition, a transition stage exists in which the adrenal medulla is not yet fully matured while the extra-adrenal chromaffin tissue (serving thus far as the major source of circulating catecholamines) has already started its postnatal involution . Such postulation could account for the marked reduction in plasma levels of norepinephrine and epinephrine in the first few days after birth . However, the unchanged levels of plasma dopamine after birth is harder to explain unless this catecholamine on mates mostly from SIF cells which do not require support by glucorticoids ~17) . The subsequent rise in plasma norepinephrine and epinephrine towards the second week of life is therefore in accordance with the delayed maturation of the adrenal medulla in the newborh rat . It has been reported by Popper et al (18), and also confirmed by us in previous studies (unpublished observat3ön7, that decapitation causes a marked elevation in plasma norepinephrine and epinephrine (but not dopamine) as com pared with their levels in plasma collected under minimal stress situations . However, because of the minute size of fetal and neonatal rats and the technical difficulties of implanting chronic indwelling cannuli in term fetuses or in nursing newborns, such a method of blood collection was not feasible in our studies . Still, the possibility should be considered that a differential response to stress situations such as handling and decapitation by fetal, newborn and adult rats may also contribute to the unequal concentrations of their plasma catecholamines .
Açknowledgements The author wishes to thank Miss Ruth E . Maxson for technical assistance and Mrs . Terra Spradlin for secretarial help . This research was supported in part by funds from Indiana Affiliate of American Heart Association and by Grant USPHS RO1 NS 13243 . The author is the recipient of a Research Career Development Award PHS K04 NS 219 . References l, 2, 3, 4, 5, 6, 7, 8, 9,
A . DOST, Pro . Horen . Res . 22 541 (1966) . R . S . COML an R,~rit . Med . Bull . 22 16 X1966) . A . V . EDWARDS, J . P . siol on on (Tf64) . N . BEN-JONATHAN an ORTER, En o~crinolo 98 14971976) . N . BEN-JONATHAN and R . E . MAXSON, n ocr no o 649 1978j, B . HOKFELT, Acta P~ siol . _Scand . upp ßf51) . C . T . JONES an~ .~0 . ROBINS~J .~h siol .~ London 248 15 (1975) . S . PARVEZ, and H . PARYEZ, Hormone as . _ 321 (1974) . G . B . WEST, D . M . SHEPHERD and A . R . MacGREGOR, Clan . Sci . 12 317 (1953f .
lag
Vol . 23, No . 1, 1978
10 .
Fatal and Neonatal Plas~m Catecholaminee
43
M . R. G. P. DeGALLARDO, F. KREIER, and J . H . TRAMEZZANI, Acta Physiol . Latinoam . 24 290 (1974) . 11 . 3~Ff.-Kb~LOGr in Nonstriatal do amine is neurons, p . 553, ed . by E . Costa and G. L . esa, aven ress, ew or 976) . 12 . M . LEM~INEN, Acta Physiol . Stand . , 62 Suppl . 231 (1964) . 13 . J . ROFFI, and A~Ô3`~~~enc~54 ßf75 (1966) . 14 . R. J . WURTMAN and J . AXELTtÖ6,Sc~iénce 150 1464 (1966) . 15 . R. D. CIARANELLO, D . JOCOBOWIT~and-J . AXELROD, J . Neurochen . _20 799 (1973) . 16 . K. M . MALINOWSKA, R. N . HARDY and P. W . NATHANIELS, Experientia 28 1366 (1972) . 17 . ~ FRANKO, and 0. FRANKO, Acta Physiol . Stand. 84 125 (1972) . 18 . C. W . POPPER, C. C. CHIUEH ahn T . -3. RbPIA~. P~rmacol . Exp. Ther . 202, 144, 1977 .
Pergamon press
PLASMA CATECHOLAMINES IN FETAL AND NEONATAL RATS Nira Ban-Jonathan Department of Physiology Indiana University School of Medicine Tndtanapolls, Indiana 46202 (Received in final form May 12, 1978) Summary During the last day of gestation, dopamine was higher in fetal than to maternal plasma whereas norepinephrtne and epinephrine were slmtlar . Immediately after birth, plasma norepinephrine and epinephrine fell to 10x of their levels in term fetuses, remained low to the second day of life and reached adult levels within one to two weeks . Plasma dopamine, however, did not reduce much after birth . The data are consistent with the predominance of the extra-adrenal chramaffin tissue in the fetus, its postnatal involution, and the delayed maturation of the adrenal medulla in the newborn .
It has been suggested that fetal catecholamines participate to the control of placental circulation ~1), and that the most potent stimulus for their release is hypoxia (2) . Epinephrine secretion 1n the newborn animal mi ht protect it from hypoglycemic reactions that sometimes occur after birth ~3) . In view of these important cardiovascular, pulmonary and metabolic functions served by catecholamlnes, it is surprising that so little information is available on their plasma levels in fetal and newborn animals . This is probably due to the lack of sensitivity of existing methods for measuring these compounds in the minute amounts of plasma obtainable from fetal and neonatal rats . Yle have recently developed a very sensitive double isotope radioenzymatic assay which 1s capable of measuring simultaneously do amine, norepinephrine and epinephrine in as little as 25-50 ul of plasma (4~ . By utilizing this assay we demonstrated that during gestation in the rat, catecholamines were compartmentalized between the fetal and maternal clrculations and the amniotic fluid ~5) . Moreover, we found that in 18 day old fetuses, dopamine constituted as much as 30~ of total plasma catecholamtnes whereas it was no more than 4-5~ of the adult's . Thus, fit became of finterest to extend these studies and examine the changes occurring in plasma catecholamtnes immediately after birth and during the first two weeks of neonatal life .
0300-9653/78/0703-0039502 .00/0 Copynght © 1978 Pergsmon Press
40
Fatal and Neonatal Plasma Catecholaminea
Vol . 23, No . 1, 1978
Materials and Methods Male and female Wistar rats, weighing 250-300 g sere used 1n this study . Rats in proestrus were placed overnight with males and the next morning, with the appearance of vaginal sperm, was designated as day one of gestation . All animals were decapitated between 10 and 12 a .m . to eliminate diurnal variations . Trunk blood was collected into chilled heparïnized tubes and the plasma separated and stored at -20 °C . Pregnant rats were decapitated ôn day 22 of gestation which is the day of parturition . The uteri were immediately removed and the fetuses decapitated . Fetal trunk blood was collected by the aid of heparinized pasteur pipette and pooled from every 2-3 fetuses to a final volume of 200 ul . The time from decapitation of the mother to collection of blood from all the fetuses did not exceed 15 minutes . No significant differences were observed between catecholamines in plasma collected from fetuses immediately after maternal decapitation or 10-12 minutes afterwards . Newborn rats were decapitated on days 1 (the day of parturition), 2, 7 and 14 of postnatal life . Trunk blood was pooled from 1-2 neonates to a final volume of 300 ul . Catecholamines assay : Triplicate aliquots (25 or 50 ul) of plasma were incubated with catechol-0-methyl transferase and S-adenosyl-L-methionine The 0-methylated dopamine, norepi(methyl- 3 H) as described previously (4) . nephrine and epinephrine were extracted, separated by two-dimensional thin layer chrromatography, and counted in Aquasol in a liquid scinttllntion spectrometer . The assay sentitivity was 5-15 pg for each amine and the overlap between arpr two of the three compounds did not exceed 1-2x . The results were test . analyzed by a student
x
Results and Discussion As shown in Fig . 1, plasma concentrations of dopamine, norepinephrine and epinephrine 1n pregnant rats on the last day of gestation were 0 .6210 .12, These values do not 3 .7510 .59 and 9 .0511 .67 ng/ml (meanslSE), respectively . differ significantly from catecholamine levels in plasma collected from decapitated male rats . Thus, there was no apparent effect of advanced pregnancy on the levels of circulating catecholamines . On day 22 of gestation, norepinephrine and epinephrine concentrations in fetal and maternal plasma were similar . Considering that at that time, the fetal adrenal content of these compounds does not exceed 1-2X of the adult's (6), such finding raises the possibility that catecholamines transfer between the maternal and fetal circulations . However, there is a dissimilarii~y in Moreover, in our prevdopamine concentration in maternal and fetal plasma . ious study (5) we found that on days 18 and 19 of gestation, epineph M ne was 5 fold lower in fetal than in maternal plasma . Other evidence against placental transfer of catecholamines exists (7), although Parvez and Parvez (8) demonstrated an uptake of tritiated epinephrine, injected into the mother, by the fetal heart . A likely explanation for the high plasma levels of catecholamines in the fetus at term is that they primarily originate from tissues other than the adrenal . Indeed, the fetus possesses a unique structure, the extra-adrenal chromaffin tissue, which reaches its maximal structural growth at birth and On day 22 of fetal life, regresses within two weeks of postnatal life (9) . the content of norepinephrine and spinephrine in this tissue equals or exceeds that of the adrenal medulla (10) . Fetal plasma dopamine might originate either from the extra-adrenal chrbmaffin tissue or from SIF (small, intensely fluorescent) cells found in structures such as the superior cervical gan lie and the carotid bodies which contain considerable amounts of dopamine ~11) .
Fatal aad Neonatal Plasma Catecholamines
Vol . 23, No . 1, 1978
+
41
aoPSIIIRi ,_.,
,~~,
II
f-1
II
II
RORiPIRiPRRIMi
ë
~~
r
,
+_
0 ~, a i O O u 1+ r H u e a lo ~ m
RPIRiPRRIRi
(.)
PARI'Oiliplf ('I)
(6)
1
(1>)
a
M~II
va
(1~
Pe4rt
(n~wa
~l
P,lt... (o
~
Ratw
~a
FIG .
n
dv)
I"I
IUm.lw
a wa
II
tlq,lw (+ ao .)
II
R.a.aw
n+ ~a
1
Plasma catacholamines at different developmental stages . Day 1 of gestation was desi nated with the presence of vaginal sperm . Day ~ of neonatal life was the day of parturition . A11 decapitations were done between 10 and 12 a .m, and trunk blood collected into heparinized tubes . Blood was pooled as follows : from 2-3 fetuses ; from 1-2 neonates and from slngle adults . Catecholamtnes were determined by a radioenzymatic assay in triplicate aliquots (25 or 50 u1) . Values are meanstSE and the number of pooled samples per group is shown in parentheses .
On days 1 and 2 of neonatal life, plasma norepinephrine and epinephrine were extra y low, compristng not more n 10~ of their levels in fet 1e plasma at term . By da~yr 7, plasma levels of these compounds increased, and by day 14 they reached or surpassed adult plasma levels . po~amine, however, dtd not fall much after parturition and was stt11 higher in plasma fr~am two week old neonates than that from mature males or pregnant finales .
42
Fetal and Neonatal Plasma Catecholaminae
Vol . 23, No . 1, 1978
Several lines of evidence indicate that glucocorticoids maintain the integrity of the extra-adrenal chromafftn tissue in fetal animals (l2, 13), as well as stimulate an increase in the activity of the norepinephrine methylating enzyme PNMT (phenylethanolamine-N-methyl transferase) both in the adrenal (14) and the extra-adrenal chromafftn tissue (15~ . The cause for the involution of the extra-adrenal chromaffin tissue immediately after birth has been attributed to the withdrawal of glucocorticoid support resulting from a reduction in their circulating levels . It has been argued that such withdrawal would not be detrimental for the maintenance of adrenal medullary cells because of their proximity to the cortex and the constant exposure to very high concentrations of glucocorticoids . Indeed, in 1966, Jost (1) demonstrated a decrease in pituitary stimulation of the adrenal cortex immediately after birth . Recently, this was supported by the finding that circulating corticosterone in the rat was reduced in the first eight hours after birth to 20~ of its levels in term fetuses (16) . Thus, it can be postulated that after parturition, a transition stage exists in which the adrenal medulla is not yet fully matured while the extra-adrenal chromaffin tissue (serving thus far as the major source of circulating catecholamines) has already started its postnatal involution . Such postulation could account for the marked reduction in plasma levels of norepinephrine and epinephrine in the first few days after birth . However, the unchanged levels of plasma dopamine after birth is harder to explain unless this catecholamine on mates mostly from SIF cells which do not require support by glucorticoids ~17) . The subsequent rise in plasma norepinephrine and epinephrine towards the second week of life is therefore in accordance with the delayed maturation of the adrenal medulla in the newborh rat . It has been reported by Popper et al (18), and also confirmed by us in previous studies (unpublished observat3ön7, that decapitation causes a marked elevation in plasma norepinephrine and epinephrine (but not dopamine) as com pared with their levels in plasma collected under minimal stress situations . However, because of the minute size of fetal and neonatal rats and the technical difficulties of implanting chronic indwelling cannuli in term fetuses or in nursing newborns, such a method of blood collection was not feasible in our studies . Still, the possibility should be considered that a differential response to stress situations such as handling and decapitation by fetal, newborn and adult rats may also contribute to the unequal concentrations of their plasma catecholamines .
Açknowledgements The author wishes to thank Miss Ruth E . Maxson for technical assistance and Mrs . Terra Spradlin for secretarial help . This research was supported in part by funds from Indiana Affiliate of American Heart Association and by Grant USPHS RO1 NS 13243 . The author is the recipient of a Research Career Development Award PHS K04 NS 219 . References l, 2, 3, 4, 5, 6, 7, 8, 9,
A . DOST, Pro . Horen . Res . 22 541 (1966) . R . S . COML an R,~rit . Med . Bull . 22 16 X1966) . A . V . EDWARDS, J . P . siol on on (Tf64) . N . BEN-JONATHAN an ORTER, En o~crinolo 98 14971976) . N . BEN-JONATHAN and R . E . MAXSON, n ocr no o 649 1978j, B . HOKFELT, Acta P~ siol . _Scand . upp ßf51) . C . T . JONES an~ .~0 . ROBINS~J .~h siol .~ London 248 15 (1975) . S . PARVEZ, and H . PARYEZ, Hormone as . _ 321 (1974) . G . B . WEST, D . M . SHEPHERD and A . R . MacGREGOR, Clan . Sci . 12 317 (1953f .
lag
Vol . 23, No . 1, 1978
10 .
Fatal and Neonatal Plas~m Catecholaminee
43
M . R. G. P. DeGALLARDO, F. KREIER, and J . H . TRAMEZZANI, Acta Physiol . Latinoam . 24 290 (1974) . 11 . 3~Ff.-Kb~LOGr in Nonstriatal do amine is neurons, p . 553, ed . by E . Costa and G. L . esa, aven ress, ew or 976) . 12 . M . LEM~INEN, Acta Physiol . Stand . , 62 Suppl . 231 (1964) . 13 . J . ROFFI, and A~Ô3`~~~enc~54 ßf75 (1966) . 14 . R. J . WURTMAN and J . AXELTtÖ6,Sc~iénce 150 1464 (1966) . 15 . R. D. CIARANELLO, D . JOCOBOWIT~and-J . AXELROD, J . Neurochen . _20 799 (1973) . 16 . K. M . MALINOWSKA, R. N . HARDY and P. W . NATHANIELS, Experientia 28 1366 (1972) . 17 . ~ FRANKO, and 0. FRANKO, Acta Physiol . Stand. 84 125 (1972) . 18 . C. W . POPPER, C. C. CHIUEH ahn T . -3. RbPIA~. P~rmacol . Exp. Ther . 202, 144, 1977 .
