Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe1 B. G. WYSE,". R. VANPETTEN, AND W. H. HARRIS^ Division of Pharmucology and Therupeutics, Faculty o f Medicine, U n i r ~ r s i t oy f Calgary. Calgary, Alta., Canada T2N 1 N 4 Keceived September 13, 1976 WYSE, 33. G., VAN PETTEN,G. R., and HARRIS,W. H. 1977. Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe. Can. J. Bhysiol. Pharmacol. 55, 1001-1006. Responses of isolated helical strips of ovine ear artery to electrical stimulation of postganglionic adrenergic neurons and exogenous agonists were studied at various stages of development from 110 days of gestation through to adulthood. Only rudimentary responses were observed at 110-1 15 days of gestation. A parallel development of responses to noradrenaline ( N A ) . serotonin, and lysine vasopressin began sometime after 110-1 15 days of gestation and continued until 13.9-137 days of gestation but there was little development of the latter responses until more than 3-5 days post partum. Development of responses to exogenous agonists was incomplete 2-3 weeks post partum. The development of postganglionic adrenergic responses lagged behind those to exogenous NA. Two to three weeks post partum the NA maximal response was one-third that of adult tissue whereas the response to 16 Hz (highest frequency used) was one-sixth that of adult tissue. The N A threshold concentration was lower in arterial strips of adult animals than it was in those of younger animals. The data suggest that development of functional postganglionic adrenergic innervation of vascular smooth muscle begins late in gestation and continues well after birth; this development is preceded by development of vascular mechanisms involved in the response to several agonists. WYSE, D. G., VAN PLTTEN,G . R. et HARRIS.W. H. 1977. Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe. Can. J. Physiol. Pharmacol. 55, 1001-1006. Les rdponses des bandes helicoidales isolees d'artkres provenant de l'oreille de mouton B la stimulation des neurones adrtnergiques postganglionnaires et aux agonistes exogknes sont ttudikes h plusieurs stades du dtveloppement, de 110 jours de gestation jusqu'h la vie adulte. Seules des rCponses rudimentaires sont observdes 2 110-1 15 jours de gestation. Un dtveloppement parallkle des rtponses a la noradrtnaline ( N A ) , B la sQotonine et B la lysine-vasopressine commence peu de temps aprts 110-115 jours de gestation et dure jusqu'i 133-1 37 jours de gestation, mais on note peu de dtveloppement de rtponses plus tardives jusqu'h plus de 3-5 jours post-partum. Le dtveloppement des rCponses ?des i agonistes exogtnes est incomplet chez l'animal nouveau-n6 de 2 B 3 semaines. Le dCveloppement des rtponses adrdnergiques postganglionnaires survient aprhs celui des reponses B la NA exogkne. Deux A trois semaines apres la naissance, la rtponse maximale 2 la NA est 4 de celle des tissus adultes alors que la rkponse B 16 Hz (frkquence la plus 6levte qui a it6 utilisee) est & de celle des tissus adultes. La concentration seuil de la N A est plus basse dans les bandes arttrielles des animaux adultes que dans celles des animaux plus jeunes. Ces r6sultats suggerent que le dtveloppement de I ' i n n e ~ a t i o nfonctisnnelle adrknergique postganglionnaire du muscle lisse vasculaire commence tard au csurs de la gestation et continue hien apres la naissance; ce ddveloppement est prCcCd6 par le dbveloppement des mtcanismes vasculaires impliques dans les rdponses ? plusieurs i agonistes. [Traduit par 1e journal]
Introduction Little is knoLVn abollt the development of peripheral adrenergic mechanisms within the ABBREVIATIONS: NA, noradrenaline; 5-HT, serotonin (5-hydroxytryptamine). 'This work was s u ~ ~ O r t e d the h'fKCC and the Alberta Heart Foundation. 2Fellowof the Canadian Heart Foundation. W R C C fellow.
vasculature during fetal development and the neonatal period. Studies to date on the development of sympathetic control of the circulation have concentrated on the intact circulation or isolated myocardium. Fetal lambs are known to have rudimentary baroreceptor and chemoreceptor reflex arcs from 0.6 of gestation (Rudolph and Heymann 19 74). Adrenergic mechanisms are first noted in the ovine myo-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
1002
CAN. J.
PWYSIOL.
PMARMACOL. VOL.
cardium at about the same stage of gestation and continue to develop for more than 3 weeks post partum (Friedman 1972; Lebowitz et a!. 1972). Previous reports from this laboratory using the chronicaIly prepared ewe, fetus, and newborn lamb show that there are clearly demarcated changes in cardiovascuIar responses to pressor amines at various stages of development (Harris and Van Petten 1975, 1976). The present experiments examine responses of a peripheral blood vessel of the developing fetal and newborn lamb and the ewe to transmural stimulation, exogenous NA, and other agonists in an attempt to clarify the mechanisms responsible for our in vivo observations. Materials and Methods Preparation of Tissues Tissue from adult animals was obtained from mature ewes of Suff olk or Suff oIk/ Finnish Landrace breeds anesthetized with either sodium pentobarbital or halothane. Fetal tissue was obtained by Caesarian section under aseptic conditions after halothane anesthesia was administered to the ewe. Newborn and older lambs were also anesthetized with halothane. A suitable length of central ear artery was exposed through a skin incision under aseptic conditions and the artery was excised after ligation at either end. A 3-cm distal segment of artery whose outside diameter appeared to be about 1 mm was selected in the ewes. A slightly shorter length of artery was taken from fetuses and lambs and this meant excision of a major portion of the artery. The outside diameters (millimetres) of ear arteries of the fetuses (0.65 & 0.13 ( 110-1 15 days) ; 0.70 rt 0.04 (133-137 days) and lambs 0.77 -c 0.06 (3-5 days); 0.78 & 0.07 (2-3 weeks)) were not significantly different but the adult vessels were significantly larger with an average outside diameter of 1.22 zk 0.04. The tissue was immediately transferred to Krebs bicarbonate solution (Wyse 1973), kept at room temperature, and' continuously gassed with 5 96 60: in oxygen. Excess fat and connective tissue were trimmed taking care not to damage the outer tunica adventitia. Helical strips were suspended between two parallel platinum wire electrodes in Krebs solution kept at 36 9 1°C and continuously bubbled with 5% C 0 2 in oxygen. Contractions were recorded isometrically via force displacement transducers. Transmural Stimulation Rectilinear pulses were delivered to the electrode through the low-output impedance (25 Q ) of a Grass model S48 stimulator. The pluse width was set at 0.5 ms. The voltage across a 142 resistance in series with the electrode and stimulator was monitored and current strength was calculated by Ohm's Law. Thus, the voltage across the 1-s2 resistance was adjusted to 0.5 V which at about 15 V potential difference across the unknown resistance of the electrode in the bath gave
55, 1977
a supramaximal current of 500 mA. The ability of this technique to depolarize postganglionic adrenergic nerve endings without direct depolarization of smooth muscle cells has been amply documented elsewhere (Paterson 1965; Vanhoutte et al. 1967; Su and Bevan 1970). The technique was verified with ovine central ear artery by demonstration that responses to both transmural stimulation and exogenous N A were blocked by the alpha-adrenergic blocking agent phentolamine whereas only responses to transmural stimulation were obliterated by the adrenergic neuron blocking agent guanethidine (unpublished observation). Procedure Tissue was obtained from five groups of animals: mature adult ewes, 2- to 3-week-old lambs, 3- to 5day-old newborn lambs, 133- to 137-day fetuses, and 110- to 115-day fetuses. The normal gestational period of sheep is 145 days and fetuses and lambs of either sex were used. Length-tension curves were obtained in each group of animals by measurement of the response M N A at several levels of passive stretch. to 6 x Optimal resting passive tension for arterial strips of each group was obtained from the length-tension curves and strips were suspended at this level of stretch for 1.5 h before experiments began. Resting tensions used in each group were as follows: adult. 1.0 g; 2- to 3week-old, 0.6 g; 3- to 5-day newborn, 0.5 g ; 133- to 137day fetus, 0.4 g; 110- to 115-day fetus, 0.3 g. A frequency-response curve to transmural stimulation was constructed by stimulating with a 30-s train at 0.5, 1, 2, 4, 8, and 16 Hz. A N A cumulative dose-response curve was constructed starting at 6 x lo-" M and proceeding in half log,, increments up to a maximal concentration of 6 x lo-* M. The NA threshold concentration was defined as that concentration which produced a minimal recordable contraction (40 mg or 2-mm pen deflection). A 5-HT cumulative doseresponse curve was constructed starting at 2.6 x lo-' M and proceeding in half loglo increments up to a maximal concentration of 7.8 x 10 ' M. Similarly, a vasopressin cumulative dose-response curve was constructed starting at 0.1 mU/ml and proceeding in half loglo increments up to a maximal concentration of 3.0 mU/ml. Bhentolamine was added to the bath in a concentration of 2.65 x 10-'M and after 15 min the NA cumulative dose-response curve was repeated in the presence of phentolamine. The pA2 for phentolamine was calculated as outlined by Horwitz et al. ( 1974). Drugs and Data The following drugs were used: I-noradrenaline bitartrate (Sigma), 5-hydroxytryptamine creatinine sulfate complex (Sigma), synthetic lysine vasopressin (Sigma), and phentolamine mesylate (Ciba). All drug concentrations (vasopressin excepted) are expressed as the final bath concentrations in moles per litre. Vasopressin concentrations are expressed in milliunits per millilitre. Means were compared by simple t-test (nonpaired) and differences considered significant when p 0.05.
Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe1 B. G. WYSE,". R. VANPETTEN, AND W. H. HARRIS^ Division of Pharmucology and Therupeutics, Faculty o f Medicine, U n i r ~ r s i t oy f Calgary. Calgary, Alta., Canada T2N 1 N 4 Keceived September 13, 1976 WYSE, 33. G., VAN PETTEN,G. R., and HARRIS,W. H. 1977. Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe. Can. J. Bhysiol. Pharmacol. 55, 1001-1006. Responses of isolated helical strips of ovine ear artery to electrical stimulation of postganglionic adrenergic neurons and exogenous agonists were studied at various stages of development from 110 days of gestation through to adulthood. Only rudimentary responses were observed at 110-1 15 days of gestation. A parallel development of responses to noradrenaline ( N A ) . serotonin, and lysine vasopressin began sometime after 110-1 15 days of gestation and continued until 13.9-137 days of gestation but there was little development of the latter responses until more than 3-5 days post partum. Development of responses to exogenous agonists was incomplete 2-3 weeks post partum. The development of postganglionic adrenergic responses lagged behind those to exogenous NA. Two to three weeks post partum the NA maximal response was one-third that of adult tissue whereas the response to 16 Hz (highest frequency used) was one-sixth that of adult tissue. The N A threshold concentration was lower in arterial strips of adult animals than it was in those of younger animals. The data suggest that development of functional postganglionic adrenergic innervation of vascular smooth muscle begins late in gestation and continues well after birth; this development is preceded by development of vascular mechanisms involved in the response to several agonists. WYSE, D. G., VAN PLTTEN,G . R. et HARRIS.W. H. 1977. Responses to electrical stimulation, noradrenaline, serotonin, and vasopressin in the isolated ear artery of the developing lamb and ewe. Can. J. Physiol. Pharmacol. 55, 1001-1006. Les rdponses des bandes helicoidales isolees d'artkres provenant de l'oreille de mouton B la stimulation des neurones adrtnergiques postganglionnaires et aux agonistes exogknes sont ttudikes h plusieurs stades du dtveloppement, de 110 jours de gestation jusqu'h la vie adulte. Seules des rCponses rudimentaires sont observdes 2 110-1 15 jours de gestation. Un dtveloppement parallkle des rtponses a la noradrtnaline ( N A ) , B la sQotonine et B la lysine-vasopressine commence peu de temps aprts 110-115 jours de gestation et dure jusqu'i 133-1 37 jours de gestation, mais on note peu de dtveloppement de rtponses plus tardives jusqu'h plus de 3-5 jours post-partum. Le dtveloppement des rCponses ?des i agonistes exogtnes est incomplet chez l'animal nouveau-n6 de 2 B 3 semaines. Le dCveloppement des rtponses adrdnergiques postganglionnaires survient aprhs celui des reponses B la NA exogkne. Deux A trois semaines apres la naissance, la rtponse maximale 2 la NA est 4 de celle des tissus adultes alors que la rkponse B 16 Hz (frkquence la plus 6levte qui a it6 utilisee) est & de celle des tissus adultes. La concentration seuil de la N A est plus basse dans les bandes arttrielles des animaux adultes que dans celles des animaux plus jeunes. Ces r6sultats suggerent que le dtveloppement de I ' i n n e ~ a t i o nfonctisnnelle adrknergique postganglionnaire du muscle lisse vasculaire commence tard au csurs de la gestation et continue hien apres la naissance; ce ddveloppement est prCcCd6 par le dbveloppement des mtcanismes vasculaires impliques dans les rdponses ? plusieurs i agonistes. [Traduit par 1e journal]
Introduction Little is knoLVn abollt the development of peripheral adrenergic mechanisms within the ABBREVIATIONS: NA, noradrenaline; 5-HT, serotonin (5-hydroxytryptamine). 'This work was s u ~ ~ O r t e d the h'fKCC and the Alberta Heart Foundation. 2Fellowof the Canadian Heart Foundation. W R C C fellow.
vasculature during fetal development and the neonatal period. Studies to date on the development of sympathetic control of the circulation have concentrated on the intact circulation or isolated myocardium. Fetal lambs are known to have rudimentary baroreceptor and chemoreceptor reflex arcs from 0.6 of gestation (Rudolph and Heymann 19 74). Adrenergic mechanisms are first noted in the ovine myo-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
1002
CAN. J.
PWYSIOL.
PMARMACOL. VOL.
cardium at about the same stage of gestation and continue to develop for more than 3 weeks post partum (Friedman 1972; Lebowitz et a!. 1972). Previous reports from this laboratory using the chronicaIly prepared ewe, fetus, and newborn lamb show that there are clearly demarcated changes in cardiovascuIar responses to pressor amines at various stages of development (Harris and Van Petten 1975, 1976). The present experiments examine responses of a peripheral blood vessel of the developing fetal and newborn lamb and the ewe to transmural stimulation, exogenous NA, and other agonists in an attempt to clarify the mechanisms responsible for our in vivo observations. Materials and Methods Preparation of Tissues Tissue from adult animals was obtained from mature ewes of Suff olk or Suff oIk/ Finnish Landrace breeds anesthetized with either sodium pentobarbital or halothane. Fetal tissue was obtained by Caesarian section under aseptic conditions after halothane anesthesia was administered to the ewe. Newborn and older lambs were also anesthetized with halothane. A suitable length of central ear artery was exposed through a skin incision under aseptic conditions and the artery was excised after ligation at either end. A 3-cm distal segment of artery whose outside diameter appeared to be about 1 mm was selected in the ewes. A slightly shorter length of artery was taken from fetuses and lambs and this meant excision of a major portion of the artery. The outside diameters (millimetres) of ear arteries of the fetuses (0.65 & 0.13 ( 110-1 15 days) ; 0.70 rt 0.04 (133-137 days) and lambs 0.77 -c 0.06 (3-5 days); 0.78 & 0.07 (2-3 weeks)) were not significantly different but the adult vessels were significantly larger with an average outside diameter of 1.22 zk 0.04. The tissue was immediately transferred to Krebs bicarbonate solution (Wyse 1973), kept at room temperature, and' continuously gassed with 5 96 60: in oxygen. Excess fat and connective tissue were trimmed taking care not to damage the outer tunica adventitia. Helical strips were suspended between two parallel platinum wire electrodes in Krebs solution kept at 36 9 1°C and continuously bubbled with 5% C 0 2 in oxygen. Contractions were recorded isometrically via force displacement transducers. Transmural Stimulation Rectilinear pulses were delivered to the electrode through the low-output impedance (25 Q ) of a Grass model S48 stimulator. The pluse width was set at 0.5 ms. The voltage across a 142 resistance in series with the electrode and stimulator was monitored and current strength was calculated by Ohm's Law. Thus, the voltage across the 1-s2 resistance was adjusted to 0.5 V which at about 15 V potential difference across the unknown resistance of the electrode in the bath gave
55, 1977
a supramaximal current of 500 mA. The ability of this technique to depolarize postganglionic adrenergic nerve endings without direct depolarization of smooth muscle cells has been amply documented elsewhere (Paterson 1965; Vanhoutte et al. 1967; Su and Bevan 1970). The technique was verified with ovine central ear artery by demonstration that responses to both transmural stimulation and exogenous N A were blocked by the alpha-adrenergic blocking agent phentolamine whereas only responses to transmural stimulation were obliterated by the adrenergic neuron blocking agent guanethidine (unpublished observation). Procedure Tissue was obtained from five groups of animals: mature adult ewes, 2- to 3-week-old lambs, 3- to 5day-old newborn lambs, 133- to 137-day fetuses, and 110- to 115-day fetuses. The normal gestational period of sheep is 145 days and fetuses and lambs of either sex were used. Length-tension curves were obtained in each group of animals by measurement of the response M N A at several levels of passive stretch. to 6 x Optimal resting passive tension for arterial strips of each group was obtained from the length-tension curves and strips were suspended at this level of stretch for 1.5 h before experiments began. Resting tensions used in each group were as follows: adult. 1.0 g; 2- to 3week-old, 0.6 g; 3- to 5-day newborn, 0.5 g ; 133- to 137day fetus, 0.4 g; 110- to 115-day fetus, 0.3 g. A frequency-response curve to transmural stimulation was constructed by stimulating with a 30-s train at 0.5, 1, 2, 4, 8, and 16 Hz. A N A cumulative dose-response curve was constructed starting at 6 x lo-" M and proceeding in half log,, increments up to a maximal concentration of 6 x lo-* M. The NA threshold concentration was defined as that concentration which produced a minimal recordable contraction (40 mg or 2-mm pen deflection). A 5-HT cumulative doseresponse curve was constructed starting at 2.6 x lo-' M and proceeding in half loglo increments up to a maximal concentration of 7.8 x 10 ' M. Similarly, a vasopressin cumulative dose-response curve was constructed starting at 0.1 mU/ml and proceeding in half loglo increments up to a maximal concentration of 3.0 mU/ml. Bhentolamine was added to the bath in a concentration of 2.65 x 10-'M and after 15 min the NA cumulative dose-response curve was repeated in the presence of phentolamine. The pA2 for phentolamine was calculated as outlined by Horwitz et al. ( 1974). Drugs and Data The following drugs were used: I-noradrenaline bitartrate (Sigma), 5-hydroxytryptamine creatinine sulfate complex (Sigma), synthetic lysine vasopressin (Sigma), and phentolamine mesylate (Ciba). All drug concentrations (vasopressin excepted) are expressed as the final bath concentrations in moles per litre. Vasopressin concentrations are expressed in milliunits per millilitre. Means were compared by simple t-test (nonpaired) and differences considered significant when p 0.05.
