University of the Pacific Scholarly Commons University of the Pacific Theses and Dissertations Graduate School 1976 The functional development of the adrenal-sympathetic nervous system in neonatal and adolescent swine Sidney Koon Hung Woo University of the Pacific Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Animal Sciences Commons Recommended Citation Woo, Sidney Koon Hung. (1976). The functional development of the adrenal-sympathetic nervous system in neonatal and adolescent swine. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/435 This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. THE FUNCTIONAL DEVELOPHENT OF THE ADRENAL­ SYMPATHETIC NERVOUS SYSTm-1 IN NEONATAL AND ADOLESCENT SWINE A 'l'hesis Presented to the Faculty of the Graduate School University of t.he Pacific ~ -- In Partial Fulfillment of the Requirements for L:he Degree Master of Science by Sidney I\. Vloo December 1976 DEDICATION The author dedicates this ·thesis to his mother, who is an infinite source of inspiration to her children. ii ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to Dr. Hubert C. Stanton for his invaluable guidance and support throughout his encouragement in this research; Dr. Donald Y. Shirachi, for his encouragement, guidance, and advice; Dr. Marvin H. Malone for his supervision and advice in the preparation of this thesis; Dr. Charles W. Roscoe and Dr. Horach H. Loh for generously agreeing to act as members of his thesis committee. The author is also grateful to Mr. Raymond L. Mueller and Dr. Harold J. Mersmann of Shell Development Company for their interest a~d ~elE i~ this research. Finally, the author wishes to express his apprecia­ tion to Mrs. Carol Sarnoff for her technical assistance in the preparation of this thesis. iii TABLE OF CONTENTS Page LIST OF TABLES .. vii LIST OF FIGURES ix Chapter I INTRODUCTION . .. 1 Statement of the Problem 1 Review of the Literature 5 Catecholamines and the Adrenal Medulla . 5 Ontogenesis of the Sympathetic N~rvous System. • . 22 Perinatal Survival of the Swine. 25 II !4ATERIALS AND METHODS. 30 ------------ - -- ------- Animals. • • . • . 30 Estimation of Medullary and Cortical Volumes . 30 Determination of Catecholamines. 32 Determination of Catecholamine Biosynthetic Enzymes Activities 34 ','· Tyrosine Hydroxylase (TH). 36 DOPA Decarboxylase (DDC) : Method 1 •.....•.. 39 DOPA Decarboxylase (DDC): Method 2 • • • • • • • • • • 39 Dopamine-B-hydroxylase (DBH) 42 Phenethanolamine-N-Methyltrans­ ferase (PNMT) . -: . 45 Biuret Method. • . 48 Estimation of Maximal Velocity (Vmax) and Michaelis Constant ( Km) • • • • • • • • • • • • • 49 Determination of Tyrosine Concen·tra·tions in Adrenal Gland and Plasma . • 49 iv v Chapter Page II Tyrosine Extraction from Adrenal Glands .. ' •..••.• 49 Tyrosine Extraction from Plasma. so Fluorometric Determination of Tyrosine Concentration •••.. so In Vitro Biosynthesis of Catecholamines -rrom Tyrosine by Adrenal Slices . 51 Amine Uptake by Adrenal Vesicles • 52 Amine Efflux from Adrenal Vesicles 58 III RESULTS •. 59 Growth of the Adrenal Gland: General Aspects • • . • • . • • • 59 Relationship Between Adrenal and Body Weight . • • 59 Growth of the Medulla in Relation- ship to the Cortex. • • . • 59 Relationship Bet1-1een Catecholamine Content and Piglet Age. 62 Ontogenic Patterns of Catecholamine Biosynthetic Enzymes. • • • . • . 69 ~-- Tyrosine Hydroxylase (TH) Activity ancf PTgleE Age~- . -: :- . ; ;- . -:- .­ DOPA Decarboxylase (DDC) Activity and Piglet Age ....•..••. 69 Dopamine-S-Hydroxylase (DBH) Activity and Piglet Age . • • • • 73 Phenethanolamine-N-Methyltransferase (PNM1') Activity and Piglet Age •. 73 Apparent Kinetic Patterns of the Catecholamine Biosynthetic Enzymes and Piglet Age. • • 73 Synthesis of Catecholamines by Adrenal Slices in vitro . • . • 78 Effect of a-Methyl-p-Tyrosine (aMpT) on the in vitro Biosynthesis of Catecholamines. • . 78 Adrenal and Plasma Tyrosine Con- centrations and Piglet Age. 80 Ontogenesis of in vitro Catechol- amine Biosynthesis. • . • • . 80 vi Chapter Page III Ontogenesis of in vitro Amine Uptake and Efflux by Isolated Adrenal Vesicles. • 83 Relationship of Amine Uptake with Piglet Age. 83 Relationship of Amine Efflux with Age. 88 IV DISCUSSION. 92 v SUMMARY 106 BIBI,IOGRAPHY. 108 LIST OF TABLES Table Page I Piglet .1-!ortality at Five Locations. 3 II Diet Compositions, Percent. 31 III Percent of Medullary Volume in Terms of Cortical Volume as Related to Species and Age .•••..•••• . 61 IV Mean Body Weights and Adrenal Gland Catecholamine (CAl Content Correlated with Piglet Age. • • . • • . • . 63 v Mean Specific Activity of Tyrosine Hydroxy­ lase·in Adrenal Homogenates Correlated with Pigl~t Age. • . • . 70 VI Mean Specific Activity of DOPA Decarboxylase in Adrenal Homogenates Correlated with __ Pigle_t age. __ • ~ ·'--··. __. • ,__ ._. ,___. __. __ 70_ VII Mean Specific Activity of Dopamine-B­ Hydroxylase in Adrenal Homogenate Cor- related with Piglet Age. 74 VIII Mean Specific Activity of Phenelthanolamine­ N-Methyltransferase in Adrenal Homogenate Correlated with Piglet Age . • . • . 74 IX Apparent Michaelis-Menten Kinetic Parameters j of Catecholamine Biosynthetic Enzymes in ' Adrenal Homogenate Correlated with Piglet Age . • • . · · · · · 77 X Mean Tyrosine Concentration in Plasma and Adrenal Glands Correlated 1>/i th Age • 82 XI Mean Catecholamine Synthesis Correla·ted with Tyrosine Concentration and Piglet Age. 84 vii viii .LIST OF TABLES l (continued) 1 Table Page XII Effect of Piglet Age on Vesicular Up- take of Epinephrine (10 min. incuba­ tion) in the Presence or Absence of Reserpine. 85 XIII Effect of Piglet Age on Vesicular Up­ take of Metaraminol (10 min. incuba­ tion) in the Presence or Absence of Reserpine. 86 XIV Slopes of Uptake Curves Correlated with Age . 89 XV Efflux (60 minutes) of Epinephrine and Metaraminol Correlated with Piglet Age_ • . • • • • . • . • . • • • . 91 LIST OF FIGURES Figure Page 1 Norepinephrine standard curve. 35 2 Epinephrine standard curve • . 35 3 Determination of optional amount of homo­ genate, incubation time, and substrate concentration for the assay of tyrosine hydroxylase. • . • ••• 37 4 Determination of optional amount of homo­ genate, incubation time and substrate concentration for the assay of DOPA decarboxylase. • • • • . •• 40 5 Determination of optional amount of homo­ gena::e, incubation time and substrate concentration for the assay of dopamine- S-hydroxylase. ... 43 -- --- --- -------- --- -- ----- -- - -- -- 6 Determination of optional amount of homo- genate, incubation time and substrate concentration for the assay of phenethanolamine-N-methyltransferase. 46 7 Elution pattern of norepinephrine and normethanephrine. ••. 53 8 Determination of optimal parameters of the amine uptake by isolated adrenal vesicles • • 55 9 Relationship between body and adrenal weights. • . • . • • 60 10 The change of norepinephrine content as a function of age. •.. 64 11 The change of epinephrine content as a function of age. • . • • . • 66 ix X Figure Page 12 The change of catecholamine content as a function of age. • • . • . 67 13 The change of epinephrine content as a function' of age. 6 8 14 The change of specific activity of tyrosine hydroxylase as a function of age • • • 71 15 The change of specific activity of DOPA decarboxylase as a function of age • • 72 16 The change of specific activity of DOPA-S- hydroxylase as a function of age. • • . 75 17 The change of specific activity of phenethanolamine-N-methyltransferase as a function of age. • . • 76. 18 In vitro inhibition of tyrosine hydroxyl- -ase- by a-methy1-E-tyrosine . • . • . 79 19 In vitro inhibition of catecholamine bio- synthesis by a-methyl-E···tyrosine. • . 81 20 Amine uptake by adrenal vesicles of 0-day- _o:J,.g swi_ne_witl:Land_wij:hQut_rE!SE!;:p_;i.ne .__ ._. _ 87 Chapter I INTRODUCTION Statement of the Problem Birth is a severe environmental change. The neonate is abruptly exposed to the harsh extrauterine environment and must seek food and shelter on its own.. Furthermore, it must maintain homeostasis successfully in order to sur­ vive. The condition of homeostasis has been defined by Cannon (1939) as an internal state of equilibrium estab:­ lished by automatic adjustmen-ts when the animal experiences -i- environmental changes.• _These internaL adjustments are_ brought about by a number of coordinated physiological and biochemical mechanisms. The development of the animal after birth is marked by complex changes. Certain periods are particularly im­ por·tant and have been defined as "critical periods" (Krecek, 1971). Two processes are important to the sur- vival of the animal during these times. The process of "adapta·tion" involves the maturation of specific physiological systems according to the needs of the neonatal animal, and is dic·tated by the external environment. The process of 1 2 "tolerance" re.nders the neonate relatively unresponsive to· a stressor as compared to the adult and protects the animal from over-responding and depleting vital resources until it is physiologically mature. An example of tolerance is the resistance to anoxia of immature neonates. The newborn pig has a high rate of mortality. The number of pigs born 114 days after conception