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This is to certify that the thesis entitled INVESTIGATION OF A POSSIBLE FUNCTION OF ACETYLCHOLINE IN THE HUMAN PLACENTA presented by Paul August Wennerberg has been accepted towards fulfillment of the requirements for Master's Jegree in Pharmacology & Toxicology Major professor L122. . ’ I? Y Michigan Siam . - ‘- UnNcmty Date August 11, 1978 0-7 639 INVESTIGATION OF A POSSIBLE FUNCTION OF ACETYLCHOLINE IN THE HUMAN PLACENTA BY Paul August Wennerberg A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology and Toxicology 1978 ABSTRACT INVESTIGATION OF A POSSIBLE FUNCTION OF ACETYLCHOLINE IN THE HUMAN PLACENTA by Paul August wennerberg The human placenta is a noninnervated organ containing high quanti- ties of acetylcholine and its synthesizing enzyme, choline acetyltrans- ferase. This ester and enzyme follow distinct fetal developmental patterns. This observation, plus its possible analogy with acetylcholine function in neural tissue, has led to the repeated speculation that acetylcholine functions in the human placenta by altering membrane permeability. It was decided to examine six areas that might lead to examples of acetylcholine involvement in human placental functioning. These were: 1) Altered phospholipid turnover, also refered to as the phospholipid effect; 2) fetal membrane permeability changes and electrical potential generation; 3) amino acid uptake into placental fragments; 4) the charac- terization of cholinergic recognition sites; 5) alteration of cGMP levels, and 6) site and amount of acetylcholine release using a bi- laterally perfused placental cotyledon. Other than the last set of experiments which were only completed through a preliminary stage, no parameters were uncovered that would lead to a clearer delineation of human placental cholinergic functioning. ACKNOWLEDGEMENTS I would like to acknowledge and thank the following who were on my guidance committee. They were Drs. Thomas Kirschbaum; Frank welsch who directed this work; and especially Theodore M. Brody and Tai Akera for providing support, technical assistance and understanding. LIST OF TABLES TABLE OF CONTENTS LIST OF FIGURES— INTRODUCTION A. General Introduction B. Placental Cholinergic System; C. Specific Background for Research Objectives 1. Phospholipid response to ACh 2. Placental membrane permeability and potential changes 3. Placental cholinergic recognition sites 4. cGMP response to ACh 5. Site of placental release of ACh MATERIALS AND METHODS A. 32Pi Incorporation into Phospholipids B. Membrane Potential Generation and Membrane Permeabi- lity Changes 1. Membrane potential generation C. Cholinergic Recognition Sites and Uptake of lac-c- Aminoisobutyric Acid into Placental Fragments- --- 1. Tissue sources and preparations 2. Amino acid uptake studies 3. Binding studies with radioactive cholinergic com- pounds a. Equilibrium dialysis studies b. Filtration assay c. Centrifugal assay d. Radiochemicals D. cGMP E. ACh Released from an Isolated Perfused Cotyledon ----- ii Page iv 20 20 24 24 25 26 26 27 28 29 29 30 TABLE OF CONTENTS (continued) Page RESULTS 33 A. Phospholipid Incorporation 33 3.1. Membrane potential generation 41 3.2. Membrane permeability to 24Na ‘ 42 C.l. Cholinergic effects on amino acid uptake----—- 45 C.2. Examination of possible placental cholinergic recognition sites 58 D. cGMP Response to ACh 69 E. ACh release from perfused placenta 74 DISCUSSION— 81 A. Phospholipid Incorporation of 32Pi 81 B.1. Membrane Potential Generation A 83 2. Membrane permeability to 24Na 84 C.l. Cholinergic effects on amino acid uptake------ 84 2. Examination of possible placental cholinergic recognition sites 86 D. cGMP Response to ACh 89 E. ACh Release from Perfused Placenta 90 SUMMARY 92 BIBLIOGRAPHY 93 iii Table 10 ll . 12 13 LIST OF TABLES Phospholipid Response in Various Tisues Percent Distribution of 32? in Polar Phospholipids of Human Term Placenta Fragments Amnion-ZANa Mbvement Chorion-ZANa Mevement Amniochorion-ZANa Mevement (3H)QNB Displacement by Cholinergic Compounds in Filtration Assay Equilibrium Dialysis of 1,000 x g Supernatant from Human Placenta Against H-QNB Centrifugal Assay with Placental Tissue and Various Cholinergic Drugs Rat Hemidiaphragm Incubation Studies with (1251)-c- Bungarotoxin Room.Temperature Centrifuged Assay Showing Nonspecific Binding Equilibrium Dialysis of 1,000 x g Supernatant from Human Placenta Against Cholinergic Agonists cGMP Levels in Human Placenta Drug Effects on cGMP in Human Placenta iv Page 36 46 47 48 62 63 64 65 67 68 72 73 Figure 10 11 12 13 LIST OF FIGURES Diagramatic representation of structural and circula- tory cross-section of human placenta Biosynthesis of phosphatidylinositol and turnover of its phosphorylinositol group Diagramatic representation of membrane perfusion and potential generation apparatus Time course of incorporation of 32Pi into chloroform- methanol soluble material One dimensional thin layer chromatogram showing sepa- ration of acidic phospholipids Two dimensional thin layer chromatogram showing sepa- ration of acidic phospholipids Nanograms 24Na movement per 30 minute period Accumulation of l4C-a-aminoisobutyric acid by human term placenta fragments Effects 0 cholinergic blocking drugs on the accumula- tion of 1 C-c-aminoisobutyric acid by human term pla- centa fragments Effects of cholinergic blocking drugs on the accumula- tion of 14C-c—aminoisobutyric acid by human term pla- centa fragments Effects of cholinergic drugs on accumulation of lac-c- aminoisobutyric acid by human term placenta fragments- Specific and nonspecific binding of (3H)QNB to rat brain homogenates as a function of the concentration of (3H)QNB in filtration assay cGMP levels Page 10 21 34 37 39 43 49 52 54 56 59 70 LIST OF FIGURES (continued) Figure 14 15 16 Diagramatic representation of high voltage electro- phoresis run showing separation of acetylcholine (ACh) and tetraethyl ammonium (TEA), choline (Ch) and tetramethyl ammonium (TMA) after one hour Choline and acetylcholine values released to maternal perfusate during a one hour bilateral perfusion- Choline and acetylcholine, values released to fetal perfusate during a one hour bilateral perfusion—--- vi Page 75 77 79 INTRODUCTION A. General Introduction The human placenta is an organ initimately associated with fetal development. All substances must pass through the placenta to gain access to the fetus. In recent times, this passage has been recognized to be both deleterious as well as beneficial. Since a large number of substances can penetrate the placenta, the term "placental barrier", except in certain specific circumstances, no longer is thought to be an acetual membrane barrier. If physical membrane barriers do not exist, perhaps there are other means by which the placenta can protect the developing embryo and fetus. One possible means, examined in this thesis, is the possible . modulating effect of acetylcholine on placental membrane permeability. The placental corpus is fetal trophoblastic tissue that greatly proliferates following implantation in the uterine wall. Trophoblastic villi, which will contain fetal vasculature, grow out of the newly formed chorionic plate into the intervillous space formed by the erosion of maternal tissue. It is into this cavity that maternal blood enters. Exchange takes place across the trophoblastic membrane. The route of a substance leaving the maternal blood would therefore be through first, the trophoblastic cells, connective tissue and then fetal endothelial cells. A diagramatic representation of maternal and fetal vasculature is shown in Figure l. A.:ouw:finme3 .momamm .z.m How .zuamuo>fi:= maaxoo: .eucooeae .H ouowam mo ousuwumnu ofimooueo mo aoqmmwauom an moosvouoom meson one .oafiouvoz ou moaaaam mo uu< mo unoauuooon .hnmouo ww>sn season as measmunv amass mo soauoomlmmouo uncueasoufio one Heusuosnum mo :oqueuaomouoou owueaoumcfin H ouowam . 8. 5.8.333 .2» . corneas tans -. 8:233 .0539: .53 ..\~flllll/. \_._..il,,: . . . . , o ‘ ..‘ r .. a. . , u» . . v I ... o 3 . I 4 pr u . l .. v . I lt.lvt'artllrk(sl..k 2 1L. .9» . aznir. , .. . .. 2... . . . (ll .. in . . .hn all}! r: B. Placental Cholinergic System Ever since the first description of large quantities of ACh-like activity, as identified by bioassay of human placenta extracts in the early 1930's (1), various speculations have been advanced concerning the question of its possible physiological significance in placental function. Possible considerations related to neurotransmission had to be ruled out because all attempts to establish the presence of nervous tissue ele- ments have failed (2). It was soon found by several investigators, and recently confirmed, that the concentration of ACh (3,4), and the enzyme which catalyzes its synthesis, choline acetyltransferase (acetyl-CoAr choline O-acetyltransferase, E.C. 2.3.1.6, ChAc) (5,6) followed a characteristic pattern during gestation. The levels of both ACh and ChAc rose from week ten and peaked toward the end of the second trimester. This was followed by a steady decline until term. ChAc has been found in human fetal membranes (7). It has now been shown by biochemical and histochemical means that all acetylcholinesterase (AChE) present in the placenta appears to be associated with erythrocytes (8). It had been speculated (9), and recently confirmed by indirect evidence (10,11), that most of the ACh appeared to be localized in the syncytiotrophoblast in a bound form. The possible presence of bound ACh in conjunction with the apparent precipitous decline in placental ACh levels during labor led early investigators (12) to the hypothesis that ACh was synthesized and stored for use during parturition. Placentas obtained during abortions and Caesarean sections had higher levels than those specimens obtained following normal labor and vaginal delivery (4,12). To date, however, there is no further evidence to support the labor-related role of ACh. There are two lines of argument against this 5 assumed function. First, peak levels of ACh synthesis and ACh tissue content occurred approximately fifteen weeks before termination of pregnancy. Cholinergic drugs have also been found to be ineffective in inducing or delaying labor. Human uterine smooth muscle at term shows little response to cholinergic drugs, making them essentially useless as oxytocic drugs (13). The hypothesis which is most commonly discussed concerning ACh function in the human placenta is based on an analogy with the ester's role in neural transmission. It is speculated that ACh alters membrane permeability as it does in neural tissue but in a much less specific manner not related to Na+ or K+ in particular (5,6,14,15). It is postu- lated that the release of ACh from.its bound storage form might enhance or inhibit the permeability and affect transport characteristics of the fetal trophoblast to ions and endogenous as well as exogenous compounds. The evidence which suggests this possible cholinergic involvement in placental functioning is the following. Nicotine has been shown to release ACh.from presynaptic terminals (16). This effect of nicotine has been interpreted to strengthen the hypothesis that ACh affects transport in the human placenta. It is well known that babies born to smoking mothers have lower birth weights (17). It is believed that there is a critical concentration of ACh required to achieve Optimal transplacental movement of nutrients (5). The excess ACh, presumably released by nicotine, would then decrease this transplacental movement. A similar result is seen in babies born of heroin-addicted mothers (18). Morphine, in addition to other actions such as producing hypoxia, de- creases the release of ACh.in the central nervous system (19) and in the peripheral nervous system (20). It appears therefore as if compounds 6 that either enhance (nicotine) or decrease (morphine) ACh release may affect fetal growth. This is consistent with the action of ACh in the neuromuscular junction where an abnormally small or large release of ACh will eventually lead to disruption of synaptic or neuroeffector trans- mission. The idea that the cholinergic system in nonrinnervated tissues may induce alterations of membrane permeability is supported by the observa- tion that ACh appears to regulate ion movement in certain nonexcitable tissues (21). An example would be the gills of a freshdwater crab, Eriocheir sinensis. The observation has also been made that the ACh- AChE system plays a role in the control of motility in spermatozoa (22). An argument in favor of the assumption that placental ACh has a function may be based on the evolutionary course of ACh existence. It is postulated that the earliest function of ACh and its associated enzymes in primitive organisms was probably the modification of the passage of various substances across cell membranes (21). With the development of different types of structural complexity of cellular membranes this role of ACh was retained by some membranes with varying degrees of specificity. In some tissues the cholinergic system was lost while others developed it to a very high degree. By the very nature of its function, the placenta is a very old organ from an evolutionary point of view. Therefore, it is conceivable that this earlier general function of ACh has been retained. 7 C. Specific Background for Research Objectives A major problem associated with placental research of this type is the lack of measurable parameters. Due to this, the specific experi- ments presented in this thesis appear to be unrelated. The purpose, behind the experimental designs, was to examine possible parameters that might show an alteration if the cholinergic system was altered. Each of these sets of experiments was conceived as an initial step. If they showed promise in revealing a relationship between the cholinergic system and placental functioning, in depth experiments in that area would have been carried out. The intent was to examine as broad a spec- trum of placental parameters as possible. The specific aspects to be examined were as follows: 1) Does ACh alter villi phbspholipid turnover?; 2) Do human fetal membranes possess the ability to generate an electrical potential iguziggg given the absence or presence of ACh and is the membrane permeability changed?; 3) Do cholinergic recognition sites exist and can their presence be asso- ciated with some function?; 4) Is a response to ACh mediated via the second messenger, cyclic guanosine monophosphate (cGMP)?: 5) Is ACh released preferentially to one circulation and what might this mean? Several approaches can be taken to investigate the role of ACh in the functioning of human placenta. Due to ethical considerations concerning human subjects an animal model would be extremely useful. Some in zigg transport studies have been done in animals (23). It would not be justifiable to use laboratory animals in the proposed experiments on the placental cholinergic system due to their lack of similarity to human placenta with respect to ACh and ChAc. While it is possible that small amounts of ChAc are present in the placentas of domestic and 8 laboratory animals (6,24), they do not reach the levels realized in human placentas and it is impossible to quantify the synthetic enzyme. There- fore, it may be inappropriate to employ an animal model and project inferences to the human. It has been found that placentas from rhesus monkeys contain ChAc activity of a magnitude similar to that of the human placenta (25,26). It appears that for a qualitative evaluation of the hypothesis of ACh involvement in placental transport, the human placenta is most suitable. If the hypothesis can be substantiated in this pre- paration, it might become warranted to proceed to ig_zigg_experiments using higher primates. l. Phospholipid response to ACh The modulating effect of ACh on phospholipid metabolism has been demonstrated in a number of nervous tissue preparations (brain and ganglia) and in a variety of glandular tissues (e.g., avian salt gland, parotid gland, pancreas) (36-40; see Table 1). In general, the addition of cholinomimetic drugs resulted in an increase in labelling from radioactive inorganic phosphorus, of phosphatidic acid (PA; 1,2-diacylglycero1 phosphate) and its derivative, phosphatidyl inositol (PI). The functional significance of this pheno- menon is still not clearly understood but the ACh stimulated turnover of PA and PI-also refered to as "the phospholipid effect"-has been impli- cated in the control of permeability in excitable membranes (37). This is possibly due to the relative rates of synthesis and degradation of one or more of the phosphoinositides, probably PI. It has been demon- strated that PI is the most metabolically active phospholipid in the sciatic and vagus nerve trunks (71). The route of turnover between PA and PI is seen in Figure 2. TABLE 1 Phospholipid Reaponse in Various Tissues Tissue Stimulating Agent Pigeon pancreas ACh or pancreozymin Submaxillary gland ACh or adrenaline Parotid gland ACh or adrenaline Peptic mucosa ACh Salt gland ACh Adrenal medulla ACh Sweat glands ACh Cerebral cortex ACh Cat stellate ganglion ACh Cat superior cervical ganglion ACh Rat superior cervical ganglion ACh Adopted from Hokin (36). The effect of most of these stimulations involved membrane permeability changes. lO .Aosv race caeeurrarora .rmuaouasotaasrausrarora .Hm .aoonm Houwmonaaauoeemone one we uo>oeusu one aouamosaahmqumaemona mo nwmonuehmoam .N ouowfim .1. N ouswam 2.2. 3335.52.92... .o .2652... \)I\lll\"l IInl'll'"lu'|\l-."II'II.IIIIIIIIU'II'uI-.IIIIIIII- 5.: A... .e. 3-. I .2350 Thu 38u:o.»ue_siou d 50¢ r813 :38 _..:o . Ail-V « u _ 95.. cu rises... .. kl? _ z a . n (a T. .12....3 \\\“"II"II|‘IIII 'II"|II'IIII' 'T'-'-"\ an: 2330 _ a h< .2332303 . ~.. r.o:3¢:to...:oit F k 12 On the basis of clinical observations, and the effects of certain pharmacological agents on fetal growth, it has been speculated repeatedly that the presence of the human placental cholinergic system may be related to permeability and transport processes across the pla- cental barrier (5,6,14). The fat content of the human placenta is comprised of 67.72 lipids with 76.62 of this being phospholipids (72). Most membranes are comprised of approximately 70% polar lipids. The formation of a phospholipid-amino acid complex has been reported to provide a means of transfer of amino acids from mother to fetus (41). It has also been shown that significant amino acid concen- tration gradients are present early in the second trimester of human pregnancy (42). The phase of rapid growth during human fetal develop- ment coincides withthe highest activity of ChAc and highest ACh concen- trations (5). It has been shown that carbamylcholine stimulated 33? uptake into PA and PI (73) in rat brain. These authors claim that 99% of the label is in PA and PI while these phospholipids comprise only 6% of the total phospholipid content by weight. It has been claimed (73,74) that ATP is not an intermediate in the stimulated pathway. Carbamylcholine or ACh does not raise the specific activity of ATP. It has also been reported (73) that there is no apparent change in the endogenous level of phospholipids and therefore the increased label incorporation is due to an increased turnover in the PA, PI cycle. It is possible that this cycling between PA and the water soluble sugar derivative, PI in the lipid bilayer opens "pores" which could have high ionic or molecular conductance. In this way, placental membrane permeability might be likened to neuronal permeability changes. l3 2. Placental membranegpermeability and potential changes Published reports are conflicting regarding the existence of an electrical potential across the human fetal membranes, the amnion and chorion, $2;E$E£B.(27928)° The characteristics of these membranes are of some importance due to the limited maternal-fetal exchange that occurs via this route. It was reported that both an electrical potential and an active polarized sodium.transport system with the direction of sodium transport from fetus to mother exists in the human fetal membranes (28). It has also been observed that sodium rapidly moves from the amniotic to the maternal fluid compartment after hypertonic saline injection (29). Electrical potentials have been recorded across the fetal membranes of animals (30,31). The rabbit allantoic membrane has been reported to contain both AChE and ChAc, and the electrical potential across the allantois was sensitive to ACh (31). 3. Placental cholinergic recognition sites Based on the possible analogy to neural tisSue and seeking to identify similarities and/or differences between innervated and non- innervated ACh containing tissues, this investigation was undertaken to determine the existence of placental cholinergic recognition sites, as defined in neuropharmacology. So far there is a total lack of func- tional parameters identified whose response to drugs can be measured as an indicator of placental cholinergic recognition site physiology. We Chase the measurement of active intracellular amino acid accumulation in fragments as a functional test to examine the possibility of a role for AKRI and cholinergic recognition sites in the human placenta, because spe- culations concerning ACh.function included regulation of active transport. 14 There are four types of preparations of human placental tissue used in experimental work. The most common are tissue slices, fragments, minces or homogenates (23). Such materials are primarily useful in gathering biochemical data. This is due to the fact that normal trans- placental movement of material from the maternal to the fetal side of the placenta involves the crossing of at least four cellular membranes. At most, the inuyiggg procedures with slices or fragments allow the measurement of intracellular accumulation of the compound under investi- gation. This is, however, only one aspect and the first step in the uptake process into the trophoblast cells preceding transplacental movement. These experiments were done initially to provide a basis for the more classical biochemical receptor labelling studies. It was observed that the uptake by human term placental fragments of 140-0- aminoisobutyric acid, a nonmetabolizable neutral amino acid, is against a concentration gradient. This uptake was significantly reduced in the presence of 1 mM atropine but not of d-tubocurarine (32). It was also reported that increased ACh depressed uptake of this amino acid (33). Further, atropine affected ACh release from floating villus (10). These observations suggested the existence of a muscarinic-type cholinergic recognition site. The existence of such a macromolecule in the placenta has been postulated and it was speculated to be nicotinic (5) although more recently incomplete experimental evidence suggested the presence of a muscarinic type cholinergic site (10,32). Attempts were made to biochemically label these sites by equilibrium dialysis or centrifugal assay with cholinergic agonists (IHPACh, 3H-nicotine), a muscarinic antagonist (3Hrquinuclidinyl benzilate) or a nicotinic antagonist 15 (lZSI-u-bungarotoxin) in subnanomolar to micromolar concentrations. There is a report which describes by means of biochemical labelling techniques and correlation with pharmacological studies on contractility the presence of muscarinic receptors in the non-innervated smooth muscle of chick amniotic membranes (35). 4. cGMP response to ACh There is considerable evidence that some muscarinic cholinergic effects are mediated via cGMP. It has been shown that ACh, and other primarily muscarinic agonists such as bethanechol, methacholine and exo- tremorine, increase cGMP levels. This has been shown in several mamma- lian tissue preparations including bovine superior cervical ganglion (43), isolated rat heart (44), rabbit cerebral cortex and guinea pig ileum (45). In addition to this, the increase in cGMP was blocked by use of the muscrainic antagonist atropine but not the nicotinic antago- nist hexamethonium. There was a question in the literature concerning the existence and type of cholinergic receptor in the human placenta. This set of experiments was undertaken in hopes of clarifying this question and to examine a possible measurable parameter associated with cholinergic function in the human placenta. 5. Site of placental release of ACh A group of experiments was planned to determine the site of release of ACh. Previous investigators suggested that the release of ACh occurs to either the fetal (52) or the maternal (5,10) circulation. None of these experiments were performed with a bilaterally perfused placenta. Therefore, at best the results are speculative. Once the location and relative amounts of released ACh are determined, l6 researchers would be better able to locate the potential site of action and function (5) of ACh. As mentioned, the above published reports did not make use of a bilaterally perfused placenta. It was therefore impossible to simul- taneously measure both the maternal and fetal vasculatures and unquestion- ably locate the side of release. This, and other requirements in pla- cental research led to the attempt to perfuse the entire placenta from both sites (46-48). Numerous problems exist regarding this technique. One major difficulty is the adequate simulation of blood flow in over 100 spiral arteries which provide perfusion of the maternal intervillous space. The other difficulty relates to obtaining adequate numbers of entirely intact placentas under normal delivery conditions. If there are breaks in the continuity of the tissue barrier, leakage between fetal and maternal circulations will occur and prohibit the use of such specimens for transport studies. An alternative approach was developed by Panigel (49) to circumvent the above drawbacks in perfusing the entire organ. This method takes advantage of the anatomy of the human placenta, which has a lobular structure made up of separate functional units called cotyle- dons. Each cotyledon has an artery, capillary bed, and vein that constitute a complete circulatory system. The isolated perfused cotyle- don is being used by several investigators (49,50), including now, in our laboratory. The probability of finding one intact unit without breaks in the membrane continuity and suitable for cannulation is higher than with the entire placenta. There is also the obvious savings of materials since one cotyledon amounts to only approximately 5% of the total placenta. To date, transport of several classes of compounds, 17 ions and amino acids (5), and fatty acids (51) has been studied using the single isolated cotyledon. The use of this preparation is the most promising method to test the hypothesis that ACh is involved in the transfer of materials from the maternal to the fetal circulation. Another problem that had to be overcome was the actual measure- ment of ACh in a balanced salt perfusate. The problems stemmed from.the following two sources. These were the very small amount of ACh with respect to a large fluid volume and the large choline to ACh molar ratio (220:1) which made it hard to separate the two compounds. This analysis proved to be extremely difficult and time consuming. Due to these problems, this set of studies could not be carried out beyond a few preliminary experiments. MATERIALS AND METHODS A. 32Pi Incorporation into Phospholipids Human term placenta deriving from vaginal deliveries or elective Caesarean sections were obtained fromltwo local hospitals and trans- ported to the laboratory in an ice chest where they were used within a few hours after delivery. The placenta was sliced at about 1/3 of its total thickness parallel to the decidua basalis exposing the functional fetal villous tissue. Small fragments were teased off with forceps and collected in ice cold modified Krebs-Henseleit medium with the following composition (final concentrations in mM): NaCl 145; KCl 4.8; MgSO 1.2; 4 CaCl2 2.5; glucose 11.1; Tris(hydroxymethyl)-aminomethane base 50 mM, adjusted to pH 7.4 with HCl. The tissue pieces were freed from blood by repeated washing in the buffer. They were blotted under well defined conditions (2.2 kg/ZO cm2 for 60 sec) and any nonfunctional, nonvillous tissue which was distinguishable by its different coloration was dis- carded. Tissue samples of 50 mg fresh weight were weighed and placed into 22xl75 mm culture tubes. The total incubation volume was 2 ml 3M ACh' iodide, 0.1 ml 2xlO-3M physostigmine consisting of: 0.1 ml x10- sulfate, 0.1 m1 2xlO-4M atropine sulfate as desired or an equivalent volume of buffer, 0.2 ml (25-30 uCi) of 32F made up in tris buffer and an additional amount of tris buffer which was required to obtain the final volume. The samples were incubated in an atmosphere of 5% CO2 in Oxygen for 90 min at 37°. At the end of this incubation period 8 m1 of 18 l9 chloroformpmethanol (1:2; v/v) were added. The tubes were transferred immediately into an ice bath and homogenized for two 30 second periods at a setting of 10 with a Polytron homogenizer (Brinkmann Instruments, westbury, N.Y.). The lipids were extracted (53) and the non-lipid containing fraction dried at 50°C under a stream of N2. The dried samples were sealed and stored at -20°C for no longer than one day prior to analysis by thin layer chromatography (TLC) on silica gel plates (Silica Gel 60, #5763, Brinkmann, westbury, N.Y.). For application to the plates the phospholipid fractions were dissolved in 200 pl chloro- form and 25 ul were spotted. The plates were developed in two dimen- sions (54) and/or in one dimension (chloroform:ethanol:glacial acetic acidzwater, 50:32:ll:3 v/v). Lipids were visualized by exposure to iodine vapors and identified by means of authentic phospholipid stan- dards from commercial sources (Supelco, Bellefonte, Pa. and Avanti Biochemicals, Birmingham, Ala.). Radioactive phospholipids were loca- lized by autoradiography on Kodak no screen X-ray film by exposure for about 18 hrs. After a spot was identified it was scraped off the plate into a counting vial and 10 ml of scintillation fluid (toluene con- taining 4 g PPO and 0.1 g POPOP/L mixed with Triton-X100; 2:1, v/v) was added. The vials were shaken for 30 min on a reciprocating shaking apparatus and 32P radioactivity was then determined with a model LS-lOO liquid scintillation spectrometer (Beckman Instruments, Fullerton, Calif.). All data were recorded as counts per minute. ACh iodide, physostigmine sulfate (Physo.) and atropine sulfate (Atrop.) were obtained~from Sigma Chemicals (St. Louis, Mo.). H3 32P04, carrier free in 0.02 M HCl was purchased from New England Nuclear (Boston, Mass.). Equal volumes of this solution were mixed with 0.02 M NaOH. Statistical 20 analyses were performed using Student's t-test for which the level of significance was selected at p<0.05. B. Membrane Potential Generation and Membrane Permeability Changes_ The equipment and basic technique was that described by Rose (55) and is shown in Figure 3. The equipment consisted of two lucite blocks which when joined together formed a 10.0 ml fluid chamber separated in the center by a membrane partition. The membrane, whose surface area equals 2.5 cm2, was inserted into the ring system.and the blocks were then screwed together. Four-and-one-half to 5.0 ml of the appropriate buffer was added to each side simultaneously to minimize the possibility of membrane rupture. A 52 CO2 in oxygen line was then attached to the air line inlet to facilitate mixing, maintenance of pH and oxygenation of buffer. This was the basic set-up for either the potential generation or permeability change experiments. 1. Membrane potential_generation The electrodes were miniature calomel electrodes (E.H. Sargent .mnd Co., Detroit, Mich.) filled with saturated NaCl instead of KCl. The electrodes were initially balanced by assembling the apparatus as it would be used with the exception of the addition of the membrane parti- tion. The recording equipment was a Grass Polygraph D.C. driver ampli- fier (Grass Medical Instruments, Quincy, Mass.) model 7DAC with a low level D.C. pre—amp model 7P1A. To initially test the procedure, frog abdominal skin was examined. The tissue was excised and placed in frog Ringer's buffered salt solution at 20°C that was previously equilibrated with 5% CO2 in oxygen. The tissue was then inserted in the apparatus and frog Ringer's 21 .uomuooou n ma mode: banana u mm “vasam you menu huo>ooou I am maoumhm mods I mm muwo>uomou n m “seaweeds I : momouuomao u m "menu hso>fiaom I an “Hosanna n no memo I 0 “mega was a A4 .Ammv msueumeeo coauouosow Hoausouoe one sowmnwuoe codename mo masseunomouaou ofiumamuwmfin .m ouswfim 23 solution was added on both sides. The electrodes were added and the entire apparatus was placed in a water bath heated to 20°C. The resul- tant potential change was then recorded. The tests investigating human membrane potential used pieces of amnion, chorion and amniochorion. Fetal membranes were obtained from either normal vaginal deliveries or elective Caesarean sections. The entire placenta or only the fetal membranes were placed in warm saline or Earles"buffer and returned to the laboratory in an insulated con- tainer. The membranes were carefully removed and placed in 37°C buffer which was pH adjusted and equilibrated with 52 CO2 in oxygen. The appropriate membrane was dissected out using the sleeve insert as a mold and then inserted carefully into the apparatus and Earle's buffer added. The entire apparatus as previously described was then placed in a 37°C waterbath. Some experiments had acetylcholine at a final concentration of lxlO-AM added to either the maternal or the fetal side. 2. 'Membrane permeability chaggeg, The membrane permeability studies used the same procedure and apparatus as above but without the electrodes. When 24Na movement was studied, a trace amounts of 24Na was added to either the maternal or fetal surface of the amnion, chorion or amniochorion. To remove a possible concentration gradient effect 23Na was added to the opposite side. Sampling was done on the side opposite 24Na addition. ACh was added to observe any possible effects of the ester on 24Na movement. In the case of 24Na, radioactivity will be determined in a Packard gamma counter at a window setting of 300-500 and a gain of 6.52. The 24Na was produced by the Department of Engineering at MSU by irradiating 0.1 M NaOH. A ten minute irradiation resulted in a specific activity of between 3 and 20 uCi/mg Na. The normal value was about 10 uCi/mg Na. 24 Adding 10 ul of the NaOH solution to the incubation set-up and gassing for five minutes led to a pH of about 7.45. C. Cholinergic Recognition Sites and Uptake of 14C-a-Aminoiso- butyric Acid into Placental Fragments 1. Tissue sources and preparations Human placenta from either uncomplicated vaginal deliveries or elective Caesarean sections were placed in an ice chest and transported to the laboratory. Within one half hour of delivery they were placed on a tray immersed in crushed ice with the maternal surface up. A trans- verse cut approximately one-third of the thickness of the placental disk was made to expose the fetal villous tissue. Small fragments (4-6 mm) were dissected free-hand and collected in ice cold Krebs-Henseleit buffer (KHB) of pH 7.4 which was equilibrated with 5% CO2 in oxygen and had the following composition (mM): NaHCO 27.2; NaCl, 118.0; K01, 3’ 4.8; KH P0 1.0; MgSOa, 1.2; CaClz, 2.5; glucose, 11.1. After thorough 2 4’ washing the fragments were freed from gross contamination with connec- tive tissue, major blood vessels and anchoring villi. These fragments were used directly for the amino acid uptake experiments, while a homo- genate was prepared for the cholinergic ligand binding studies. In the filtration assay a 1:10 (w/v) homogenate was made in 0.32 M sucrose. In the equilibrium dialysis and centrifugal experiments the tissue was homogenized 1:5 (w/v) in KHB containing no cholinesterase inhibitor except where noted. In all cases the homogenization of the washed fragments was achieved with a Polytron homogenizer (Brinkmann Instru- ments, Westbury, N.Y.) at a setting of 10 for two 30 second periods during which the homogenizing container was submerged into an ice bath. 25 Fractions obtained by differential centrifugation (pellet 1 [Pl] 1,000 x g, 10 min and supernatant [SNl]; the Sn was used in all cholinergic l ligand binding studies. This fraction was used without further treat- ment to remove endogenous ACh except when 3H-ACh binding was studied (see below). Upon differential centrifugation of a human placental homogenate ACh.was recovered primarily in the soluble cell fraction, however, about 602 of the ester was lost in the absence of cholinesterase inhibitor (56). Protein determinations were performed with the biuret reagent using bovine serum albumin as the standard (57). 2. Amino acid uptake studies Three solutions were made for these experiments. Solution one was KHB; solution two was made from solution one and contained inulin (methoxy-BH) 100 nCi/ml to label the extracellular water space (ECW). All drug were dissolved in solution two. Solution three contained 125 nCi/ml 14C-AIB and unlabelled AIB (500 uM) dissolved in solution two. This was diluted 1:5 in the final incubation mixture resulting in 100 3H-inulin and 25 nCi/ml l4 nCi/ml C-AIB (100 uM). Six placenta fragments ‘weighing approximately 100 mg were transferred into 25 m1 Erlenmeyer flasks containing 3.0 m1 of solution two. One ml of solution two with <3r without drug was added. The room atmosphere was displaced with 5% (302 in oxygen, the flasks sealed and placed in a water bath at 37°C for £1 preincubation period lasting up to 120 minutes. Drugs were present zoom .so«uoueeom owneeumoueaoueu Momma sans nouns mmaeeaonemone space one :« mouo>ooea hua>fiuocoavea asuou can no owousoouoe one use ozone some .moonuoz ea confluence we possesses use meanders .mouoouuxo ouo3.evfieaaoee loose oeu one comm as sea om now commenced owes Awe any enemas as nausea H.oa~.~ s.oa~.~ ~.oao.a otanom senatornooem ~.~ao.r~ s.anr.m~ m.nam.m~ Honaoooe Hanaoononorm s.oan.oa a.oas.oa o.onm.oa nao< oanaoosnnorm H.ona.~ ~.oa~.~ rc.onr.a oceanaonnroe Hsoaooennorn o.a«m.sm r.aam.~m s.anm.om naroaoou m.oan.m m.onm.m a.onr.s nanoseomnarnm ~.Hne.ms r.oar.- o.nso.~a nanoaooaoosn muoa .nono< sues .onsra cs .oosra on eo< Hononoo nenaaornnone s- on once nor me «a as mueoewoum eueoooam apes ones: we mefiafiaoeeeoem umaom ca mwm mo soauseauumaa unoouom N MAm50weanam n m usueufiuoaomma n N mofiwfiuo n H .msoaufimsoo Hmusoefiuoexo sow moaned: one nauseous: mom .mmaeeaonamoee cameos mo soaumueoom mafiaosm amHmOuoaoueo Hosea menu Hmsoamooaam 038 .e ounwwm 41 part (probably less than 5%) of the total placental phospholipid (70), PA and PI contains about 45% of the total 32Pi incorporated into polar phospholipids. The fraction of 32Pi incorporated into the identified phospholipids under the influence of drugs is shown in Table 2, columns #2 and #3. Physostigmine was used in the presence of ACh as a cholinesterase inhi- bitor to prevent enzymatic hydrolysis by placental or red blood cell cholinesterases. In some experiments carbamylcholine (carbachol), a synthetic analogue of ACh, was used. Those results were similar to that of ACh. It is not attacked by cholinesterase and no physostigmine was used. It can be seen that there was no significant change in the percen- tage of radioactivity incorporated into any of the seven identified phospholipids. Column #3 in Table 2 shows the results following treat- ment with ACh.and physostigmine combined with atropine, a drug that blocks muscarinic cholinergic receptors. Atropine inhibited the ACh 32Pi caused "phopholipid effect" in all tissues where the enhanced incorporation has been observed (36,38,40), but had no action in the placenta. Seventy to eighty percent of the applied radioactivity could be accounted for in the scraped samples. 3.1. Membrane potentialigggeration In order to test the equipment, trial runs were made using frog abdominal skin in the place of human fetal membranes. The results obtained were variable. We recorded a millivolt change ranging from 0- 30 hilliyolts within the first ten to fifteen minutes. While this is a low‘value it was consistent with another laboratory using frogs from the same source. There are seasonal factors that influence the ability to generate a potential. The inside of the frog skin was positive relative 5° the outside. 42 After this work, we felt that the system and technique was sufficiently reproducible to examine fetal membranes. In total, six placentas were examined. A minimum of three and a maximum of five membrane portions from each were examined. The multiple testing was done in response to the only literature report of a human membrane potential (28). They reported a mosaic effect where some portions generated a potential and some did not. After examining the amnion, chorion, and amniochorion, I was unable to reproduce their findings. The greatest potential that I could record was only about 1.5 to 2.0 millivolts and this might have been an artifact. During initial experiments we thought a potential was being generated but it was discovered to be a temperature artifact that occurred when the electrodes and chamber were being equilibrated. Acetylcholine was added in a final concentration of 1x10-4M to see if this might have any effect on potential generation. This was added to either the maternal or fetal sides of the amnion, chorion on amniochorion. No response was seen at times up to twenty minutes later. Due to the design of the chamber, mixing would be assumed to be vir- tually instantaneous. The fetal membranes would be assumed to be still ‘riable due to the favorable incubation conditions and the care taken in «collecting and processing the samples. l 3.2. Membrane permeability to 24Na An initial experiment was done to determine the linearity of 24Na movement over time. The results are shown in Figure 7. The move- ment was linear although somewhat declining up to at least 180 min so an experimental time course of 120 min was established. 43 tenuous sow apogee: one maewuoumz mom .moauoe ounces on use usoao>oe oz em .meoeuamsoo Houses mesumospz .m seamen 44 0mm 5 seamen mMPDZ=2 CNN 9m 09 cm on 45 A total of 17 experiments were run using the amnion, chorion and amniochorion. Acetylcholine was added to a final concentration of lxlO-AM to the side of addition of 24Na. The results are seen in Table 3, 4 and 5. Of these only three were significant. In each case, the ACh treatment lowered the amount of 24Na moved. There appeared to be a tendency for this to happen in most experiments. The significant chorion experiment probably was the result of one of the four values being much larger than the others. The other two experiments, however, clearly showed the decline. Due to the differences in both the type of membrane and site of ACh and 24Na addition, and the lack of reproduci- bility it is very difficult to interpret. These two experimental pro- cedures would be moving sodium in opposite, antagonistic directions. Specifically, the amnion experiment would involve moving sodium out of the amniotic fluid compartment while the amniochorion experiment would involve moving sodium in. 0.1. Cholinergic effects on amino acid uptake Initially, it was found that when fragments of human term placenta were incubated with 100 uMl(14C)aminoisobutyric acid (AIB), radioactivity accumulated in the intercellular water compartment against a concentration gradient in a time-dependent manner. This is seen in Figure 8. The intracellular radioactivity exceeded that in the incuba- tion medium within one hour and rose to levels six times higher over a Period of three hours. Preincubation of the placental tissue at 37°C in Krebs-Henseleit buffer (KHB) increased the concentrating ability several- f01d, depending on the duration of the preincubation as reported by other investigators (58,81) (Figure 8). 46 .mo=Ho> Houusoo scum sauceofimacmam acumen anemones so goes moxuoa mosae> .mo.ona sues once u usoooum Haouiosu a no women as: eosooaufiswfim .mz«« one :04 soon now uses mp3 soauaooo no mean any .noasoe Amusoewom .na av unusuoouu use: oso n he nosoaaow Amusoswom .mH .ov use: moo pea ooeuoe Houusoo sea Hmn.a q mH.mn«m~.mn~ a mm.mqemm.mam Hmuom m som~.q a wo.nmamm.mmm q sn.m~emw.ncm Hooch N o mm.moe q.omm N¢H.N o an.o~enc.mac assumes: H u usoauoouaxmmHQEMm Aucoauoouu menu Houuoouv souuqom< yonasz opossum mo neeaoz .a.m H mp.ozq~ an «0 seem macaquoexm someo>o= ozqmlsoasa< m mandfi 47 .oosooewuewam Hooaumaueum mouoooe xmfiuoum< .mo.ora rues some a opossum Hanulosu a no women no: ousooawacwam .ozou oso.eo< soon you uses no: soeuamoo mo page may .ooauoe Amusoawem .ma .qv usoauoouu use: one c an nosoaaow Assessmom .ma .cv use: one no: moauwe Houusoo any aoc.a o m.ams H.Hm~ o m.eme H.~om awash Ha Ham.o o m.~ma m.mmH a H.mae m.mna Hausa ca omo.a a oe.amamw.oma q om.o Hmn.mMH Hosanna: m mna.m o Hm.omnma.nma e Hm.mae H.mma assumes: a mun.a q m<.omemm.ema q mm.e~amo.qoa awesome: 5 Hem.o m mm.naa m.a~a e ~.qmawa.oma anemone: c «~.m o «o.HHH o.oma o mo.-amm.oha gaseous: m mam.o o NH.¢HH n.eHH q mn.~an n.aHH Housman: c u assausoHH\ooHaeem Ausoauoouu none HomosoUV soaufino< monasz unscrew mo uoeenz .n.m «.mp.o2¢m me no seam unoafiuoaxm ll‘l usoao>oz oz<~n:0Huer e HAmoz quNInoHuonoowsa< n mamdfi 49 Figure 8. Accumulation of 14C-o-aminoisobutyric acid by human term placenta fragments. Fragments (100 mg wet weight) were incubated in 100 M lac-AIB without (Q) or with 30 min (A) and 120 min (I) preincubation at 37°C for the time periods indicated. Intracellular radioactivity was calculated and expressed in relation to that of the incubation medium (Ratio C1/Co). Each point represents the mean i S.D. of four determinations from one placenta. RATIO ci/co 20 IS 50 INCUBATION Figure 8 I 0min A 30 min Ol20min PREINCUBATION l 2 TIME (hours) L 51 A study was then undertaken to test the effects of drugs which act on muscarinic and/or nicotinic cholinergic recognition sites as defined in innervated tissues. The compounds were examined with both nonpreincubated tissue and with fragments which had been preincubated for up to 120 minutes. In some experiments the drug and (14C)AIB were added simultaneously, while in others the drug was allowed to act on the tissue for 30 minutes prior to addition of the labelled substrate. The most consistent effect on AIB accumulation was observed when atropine sulfate (1 mM) was present. This led to a significant reduction of the concentration ratios regardless of whether the tissue had been preincu- bated or not (Figure 9). Although the drug concentration was high using criteria of neuropharmacology, the inhibitory effect was peculiar for atropine sulfate because d-tubocurarine (Figure 9) in equimolar concen- trations did not affect (;40)AIB accumulation. Atropine methyl bromide caused significant reductions of the amino acid uptake only in some of the specimens investigated (two out of six). It was also examined ‘whether the 502- ion could have been responsible for the lower concen- tration ratios, but Na SO4 (1 mM) was without effect (Figure 10). 2 Other drugs examined included decamethonium bromide, scopol- ‘amine hydrochloride, sodium sulfate, and paraoxon. These results are shown on Figure 10. The concentration range examined was 1x10"3 to lxlO-SM. No significant effects were seen except with paraoxon at concentrations of lxlO-SM at incubation times of over two hours. Acetylcholine iodide, muscarine chloride, nicotine salicylate 3 and PhySostigmine sulfate at 1x10- to leO-SM were also examined. The results are seen in Figure 11. No significant results were obtained. 52 .Amsoe mHHoov mooHo> Houusoo Scam Amo.ona V mHuoooHMH:MHm HoMMHn onsouoo no euHa moxuoa mosHo> .oucoooHe oso Boom onHuosHanouov snow mo .n.m H sooa onu ounooouaou usHoe comm .z©IOHxH u o moo ZmIOHxH n o "zeIOHNH a e “EMIOHxH n o “ouoz Amuse nouuonv onHuoHno osHuouooonsulm one Mouse nonouoev ooHEoue Hanuoa osHaouuo .Amuoe cooov oquHoo oefieouuo mo :oHuosusoosoo mean A o\Ho OHuomv aanoE sOHuonsosH one :H mono cu :oHuoHos :H oomoouaxo moo nouoHsoHoo oo3 huH>HuoooHeoa soHsHHooouusH .mH Homecoo aouu Amo.ouev hHucoonchHo nommHn meuouoo no sea: moxuoe mosHo> .wouo nooo :uHs ozone oH osHo> Hopscoo ouoHueoueoo one .oousoooHe Houo>oo Boom o:0HuooHeuouoo snow no can no .n.m « cooa onu oucoooueou usHoa zoom .on Houuaoo moo zmIOHxH n v m: IOHXH a o A: IOHxN u n “EMIOHNH I o "ouos Amy soxoasoe moo Ammv omHuoHnooumhn ocHaoHo ooo .Amnv on Some eoHconuoEoooo .Ammv ouomHoo eaHooo mo oOHuouusooooo wean .Aoo\Ho OHuomv esHooe coHuoenosH one :H none on :OHuoHou :H nooooaexo moo mouoHooHoo no: huH>HuoooHoou uoHoHHooouuoH .oooHe xoou soHuoe IsosHoue o: osons noxoouoe now uoooxo :OHuoenosH oussHa on some Amsun equ sHa cm + :Ha omv :Ha omH now nouoesosHoue ouoB Auanos no: we OOHV ousoamoum .ousoawouw ousoooHe anon noes: he mwwm oHuhunnomHosHaoIolqu mo sOHuoHnesooo one so ownue waquoHn UstonHHono mo muoouwm .oH our 55 \ ms: 20:51:02. on. .8. too be .8 .m n. n_nu “any .unv n_nv .EE a. on onnmaa .mn. raw nu m=u nu _imw mun. nu mw nu NP 0/ mgr Ava 56 .oonHo> Houuooo aouu Amo.onav mHusooHMchHm noHMHv onuouoo so euHs moxuoa oooHo> .ousoooHa ouo scum o:0Huo:Hauouoo snow «0 .n.m H cooe one ouoomoueou uoHoa zoom .on Houusoo one .2 lonm n n was a: IOHxH u o u=¢IOme n e mzmIonH u o homo: Ammv oquHso osHEmHumoohee . mzv ouonoHHom owHuooHs .Aozv ovHHOHeo onHuoomne .A:oHuoo0Hoos uoHnHHooouueH .soHuoenocH ounoHa on core Axon mH .woum :uHs on + oav mouacHa ONH mos sOHuoeoosHoum .oueoewosu ouooooHo anon soar: he nHoo oHuauoeooHooHeoloIocH mo eoHuoHnesooo so owsuv onuoeHHozo mo ouoowmm .HH oustm 57 we. wz 113d] HH onnmna Us. :U< mp 0N 58 The results shown are representative of all the experiments done. The results were the same but absolute values changed due to different preincubation and incubation conditions. Occasionally one of the above drugs would elicit a significant response but there was never any consistent trends established, except where noted above. C.2. Examination of possible placental cholinergic recognition sites Few attempts have been made to biochemically characterize cholinergic recognition sites (receptors) in noninnervated tissues. This has been accomplished by means of labelled drugs (35,84) which have been successfully applied to innervated structures for the identification of cholinergic receptor macromolecules. It was unclear whether any comparable sites might exist in the human placenta and whether they would fit the criteria of muscarinic or nicotinic sites as established in nervous tissue. Therefore, drug binding studies were initiated with the most widely used muscarinic and nicotinic cholinergic antagonists (3H)quinuclidinyl benzilate (ONE) and (1251)a-bungarotoxin (BT), re- spectively. Both compounds were available at high specific activities thus allowing their use in the low nM concentration range. When (3H)QNB was incubated with a rat brain synaptosomal fraction under conditions comparable to the ones described by Yamamura and Snyder (78), specific binding of the compound was observed which was quite similar except that the binding was maximal at about 2 nM instead 0f 4 nM that they reported. This was, however, closer to the value of 1 HM described by Heilbronn (77). This is seen in Figure 12. In this filtration binding assay, (3H)QNB (0.1 nM to 3 uM) was bound by the Placenta material. However, parallel incubations in the presence of 5 9. .msoHuHmnoo HounoBHuoaxo now moonuoz moo oHoHuouoz oom .zomwo :oHuouuHHm :H mzcammv mo eoHuouusoocoo ozu mo :oHuocsm o no mouosowgo: 53.3 no.» cu mchva mo meiosi— A.v 338930: was HOV oHMHooem .NH ousmfim 6O {O a 1 l L l . =9. 8. :2 8. 3 NIHlOHd 6w punoq aNo (Hewd 5 4 3 -9 ,x10 M H ONE) (3 Figure 12 61 competing unlabelled QNB or other muscarinic antagonists such as scopol- amine and atropine and muscarinic agonists (muscarine, carbamylcholine and oxotremorine) in 3 100-3000 fold molar excess revealed that the binding of (3H)QNB remained the same. This was regardless of the pre- sence of drugs known to compete with the labelled drug in innervated tissues. A representative group of data reflects this on Table 6. Comparable results were obtained in equilibrium dialysis assays where the amount of radioactivity bound was directly related to the (3H)QNB concentration, but independent of the presence of large excesses of unlabelled QNB or the cholinergic agonist, carbamylcholine (Table 7). These observations suggest nonspecific adsorption of (BH)QNB to placental constituents with extremely high capacity for binding of the drug and low affinity which appeared to be unrelated to the high affinity expected for cholinergic recognition sites characteristic of tissues with cholinergic innervations. Quite similar results were obtained with (1251)a-BT which was examined in the concentration range of 1-15 nM under control conditions and in the presence of 1 uM unlabelled toxin with the centrifugal assay. This assay was also used to examine (3H)QNB binding in the range of 3.3 to 100 nM, (3H)ACh.in the range of 20 to 200 nM, and (3H)nicotine salicylate in the range of 4 to 40 nM (Table 8). While (125I)c-BT binding to intact rat hemi-diaphragms, a classical nicotinic cholinergic preparation, was blocked by 67% when the tissue was preincubated with 12 uMZc-BT (Table 9) comparable pretreatment of protein derived from the human placenta failed to alter the amount of (lzsI)c-BT bound. A 62 .o:0Hancoo HoucoaHuooxo sou ovonuox moo oHoHuouoz oom .nosHeoxo ouos nounoooHe one .osOHuoeHeuouon ouooHHesn Boom .n.m H N .a.a.o ousoooueon moon .ouoo zooms sOHuouuHHm mo ouHooeeoo o>Huousomoaeom menace masses -Hrom release answer sesame Hmnrnm sarcasm season emaorm assorm unease zesoaxa ousnuo canoes omnrae zeuoaam Nmaroo amnmrm amamco canoes saucers answer ashram rwsoam cmnoom smuoaxm Azauoaxmr.ov :OHuouucoosoo .1 osHHoeonaoesou oeHuoaouuoxo oeHaoHoeoom ocHeouu< wouHuooaoo mzoammv >ouo< coHuouuHHm eH overca5oo onuosHHoeo an unoaoooHeoHn mzcammV o MHmdh 63 TABLE 7 Equilibrium Dialysis of 1,000 x g Supernatant from Human Placenta Against H-QNB Competing dpm/0.l ml Drug Drug Bag Bath _____ 473 284 3 r 33 r 8 HPQNB (0'4 nM) Carbachol 479 288 (1 mM) i 28 i 13 ______-__ 20,446 13,045 3 £1,388 2 302 HPQNB (2° “'0 QNB 20,663 13,175 (2 uM) £1,222 r 265 Duplicate samples of supernatant 1 (lo-15 mg protein per ml) were dialyzed for 24 hrs at 4°C (against 100 volumes of the drug specified. Parallel assays were run in the presence of the competing drug in the concentrations indicated. Values show the mean t S.D. of six 0.1 m1 samples removed from‘bags and baths and duplicate assays and are expressed in dis- integrations per min. 64 TABLE 8 Centrifugal Assay with Placental Tissue and Various Cholinergic Drugs Pellet Radioactivity (c.p.m.) 35mm . 3.3 nM 3H-QNB 747.25: 16.89 3.3 nM + 330 nM QNB 747.3 : 0.89 10.0 on 3H-QNB 2209.05: 44.33 10.0 nM + 1 on QNB 2103.75:100.62 100.0 nu 3H-QNB 20822.3 :817.56 100.0 nM + 10 on QNB 21110.35:546.53 3H-ACh 20.0 nM.3H-ACh 149.35: 2.61 20.0 nM1+ 20 nM ACh 201.15: 12.51 200.0 nu 3H-ACh 229.8 : 10.6 200.0 nM + 200 uM ACh 247.15: 1.35 3H-Nicotine 4.0 on 3H-Nicotine 154.85: 3.04 4.0 nM + 4 uM Nicotine 192.85: 2.61 40.0 nM 3H—Nicotine 270.85: 23.12 40.0 nM + 40 uM Nicotine 284.25: 14.92 125I-c-Bungarotoxin (BT) 1.0 nM lzsrosr 3328.0 :666.94 1.0 nM.+ 1 on cBT 3191.9 :411.25 2.78 nM 1251681 8577.4 : 49.19 2.78 nM‘+ 1 uM cBT 8596.0 :171.66 11.12 anlzschT 45285.15:175.49 11.12 nM.+ 1 uM.cBT 44499.6 :581.03 See Materials and Methods section for exerimental details. Values represent mean t S.D. from duplicate determinants. 65 TABLE 9 Rat Hemidiaphragm Incubation Studies with ( 251)-a-Bungarotoxin Dru Competing Radioactivity 8 Drug (cpm/diaphragm) 125 nM (1251)a-81 44624.75:6104.94 12 uM,aBT 29711.0 i5734.0 Preincubation with lxlO-aM KI 125 nM (1251)c-BT 23736.33:1477.85 12 uM aBT l3615.66:2378.1 See Materials and Methods for experimental details. Values are mean i S.D. for tripli- cate determinants. Each control and treat- ment was paired. 66 possible reason for only 67% being blocked in the rat hemi-diaphragm preparation and none being blocked in the placental experiment was nonspecific iodine binding. Rat bani-diaphragms were first incubated in 1x104M potassium iodide and then with (125 I)a-BT. Table 9 shows that this did not effect the percentage blocked. This unspecific (IZSI)c-BT binding was related to the toxin concentration, and when applied in a fixed concentration with variable amounts of placental protein, showed no signs of saturation of the nonspecific adsorption sites. This is seen in Table 10. In order to rule out the possibility that the low temperature of the equilibrium dialysis conditions when examining (3H)QNB and (1251)a-BT might have affected drug binding, some studies were performed under room temperature conditions with a centrifugal assay. Again, all the binding which occurred to the protein pelleted after centrifugation and repeated washings was found to be uninfluenced by competing drugs (Table 10). Additional experiments were done with agonists. Experience, however, from many receptor labelling studies has shown that only a few agonists were suitable to label their own receptors because their binding affinities were generally much lower than those of the antago- nists. In the concentration required for binding, unspecific attachment becomes a major problem (84). No specific binding could be established with.either ACh.ar nicotine (Table 11). There were no significant differences in the amounts of radioactivity bound in the presence of competing unlabelled drug in the case of ACh and nicotine did not bind at all. Room Temperature Centrifuged Assay Showing Nonspecific Binding 67 TABLE 10 Competing Pellet Drug Drug mg Protein Radioactivity (cpm) 125 2.78 nM 1631' 1.8 2992.75:158.80 1 “M (131' 2787.7 : 65.19 2.78 nM 1251681: 3.6 4919.85:169.49 1 m 013T 4603.8 :392.58 2.78 nM 1251881 6.0 7054.6 : 90.07 1 m 031' 6832.15:277.96 2.78 nM 1251681 8.4 8991.4 :206.2 1 11M 63'! 8808.75: 4.47 2.78 nM 1251881 10.8 10541.1 : 62.92 1 HM 0431‘ 10746.0 :255.26 See Materials and Methods for experimental details. Values are mean i S.D. of duplicate determinations. 68 TABLE 11 Equilibrium Dialysis of 1,000 x g Supernatant from Human Placenta Against Cholinergic Agonists Competing dun/0.1 ml Drug Drug Bag Bath 334ACh ------- 252:12 164: 13 (20 nM ACh (20 HM) 24S: 13 169: 8 3H_ACh ------ 2,430: 48 2,154: 13 (200 nM) ACh (0.2 mM) 2,494: 63 2,191: 54 BH-ACh ---—--- 12,763:124 10,718:368 (1 HM) ACh (2 mM) 12,548:261 10,529:278 3H—Nicotine ------ 177: 12 163: 13 (4 nM) Nicotine (5 HM) 16S: 44 172: 21 Carbachol (0.1 mM) 181: 10 158: 18 ««««« 1,734: 21 1,808: 60 3H-Nicotine Nfggtigg 1,747: 18 1,817: 41 (40 nM) . Carbachol (0.1 mM) 1,730: 45 1.836: 30 Duplicate samples of supernatant 1 (10-15 mg protein per ml) were dialyzed for 24 hrs at 4°C against 100 volumes of the drug specified. Parallel assays were run in the presence of the competing drug in the concentrations indicated. Values show mean : S.D. of six 0.1 ml samples removed from bags and baths of duplicate assays and are expressed in disintegrations per min. 69 D. cGMP response to ACh There were several preliminary experiments necessary to be completed before the effects of.ACh on cGMP levels could be determined. It was first necessary to determine whether the standard curve should be run in the presence of tissue and if it should be extracted. The results are seen in Figure 13. When the tissue plus extraction values had the endogenous tissue values for cGMP subtracted, it coincided with the no tissue extracted line. Therefore, in all subsequent experiments the standard curve was run through the same extraction procedure as the tissue samples. It was necessary to determine if the substance being measured was actually cGMP. This was done in two ways as seen in Table 12. Phosphodiesterase was obtained from Dr. James Bennett (MSU) and added to incubation tubes containing a set amount of tissue. The percentage of unlabelled cGMP bound, the endogenous material, dropped to almost blank values. This indicated that we were measuring cGMP. An indirect experiment, showed that as the amoung of tissue increased, the amount of cGMP present also increased. From these experiments we decided to use a tissue amount of 100 mg per sample for the remaining experiments. A total of seven experiments were done. In addition to looking at ACh, carbamylcholine, atropine and nicotine were also examined (Table 13). The final concentration of each drug was lxlO-AM. Physostigmine was used in addition to ACh. There are several time points which could have been pooled. It was decided not to do this because of the relatively large inter-experiment variability. Occa- sionally there was large variability within and between replicate pairs so a randomized complete block analysis of variance was used to remove 70 .ouocoooum :owuomuuxo wofiomuomso sonu was m>uoo vumvomum one ou compo ma mommfiu ma OOH sons Auso uncommon Amv .ouommooue oowuomuuxo mnu wofiomuoooo uos moods o>u=o unmeasum on sons mHo>mH mzoo .ma ouowfim 71 % TRACEBINDING 3/8 (100) O O O to v 00 8 l l l I 1O pmc GMP Figure 13 72 TABLE 12 cGMP Levels in Human Placenta Z Labelled Treatment c.p.m. cGMP Bound pmoles cGMP Blank 1261 1258 100 0 100 mg Tissue 469 479 28.7 2.2 100 mg Tissue + Phosphodiesterase 1209 1215 96 0.1 200 mg Tissue 367 369 19 3.9 300 mg Tissue 307 319 14.1 5.8 (estimated) Duplicate samples from one experiment. See Results section, part 4, for description. 73 .no.o weave m sues ouoeuum> mo mwmmaeom xooan ouoaaaoo wouasooomu m moan: cosmoqmqsmfim mouoson s .:o< cu coauumvm sq usomouo mm: mofiswfiumom>nm .2 oaxa no: coaumuuomoaoo mono Hosea may .oswuoofis I oaz .msfiaouum I douu< .msfiaonoamamnumo e a some .ocaaonoahuooe I so< .ooemau we oH\szo an we come uooaumouu\aouuooo nouosov M\x cc.H\Hw. om.\w~. do. \on. NB. \Nw. .oa he.a\mo.a 0H.H\HH.N mn.\mm. ca. \Hm. mm. \ H.H .oo o~.H\mm.a mm.a\oh.a HH.H\¢~.H Hm.\nm.a we.H\om.H mm. \ee.a m.a\mm.a .om oo.a\se.a ee.a\me.o .mo «Ho.a\nn.~ No.a\mn.a .NH mm.a\m~.m . HN.H\~m.H .m co.a\oc.a mn.H\mm.H me.H\H¢.H aea.\nm.a mm.a\oa.~ em. \mm.a w.H\o.~ .o oo.a\mm.~ HH.~\mH.~ n~.H\mo.~ e¢.\mm. NH.~\eH.N H~.H\nm.a n.H\m.~ .m ofiz eouu< sumo memo 50< 30< £04 noun sowusaousH a e n .4 m N H no 98:. a usmawnmaxm mueeomam seas: ca mzuo so museums mono MA Mdmdfi 74 this. When this was done, only two time points were significant. This table shows a relative decrease of cGMP with time as was seen in several of the experiments. E. ACh release from perfused placenta It was found that a necessary step that had to be incorporated into the ACh work-up was the separation of ACh and choline (Ch) using high voltage electrophoresis (EVE). An example of a typical 1 hour run is shown on Figure 14. The reason for this step is the high molar ratio difference between ACh and Ch. This value usually was about 20:1 (Ch:ACh) but ranged as high as 47:1 in one experiment. EVE was the only way to achieve satisfactory separation. Four placentas were analyzed for ACh and Ch in both maternal and fetal perfusates. Figure 15 shows the results from.a bilateral perfusion experiment examining maternal perfusate. Figure 16 shows fetal ACh and Ch release from the same experiment. Assuming a constant rate of release of 15 nm/ml for maternal Ch and 700 pm/ml for maternal ACh, 5.4 nm.Ch and 252 nm.ACh.were released during that hour into the maternal circulation. Assuming a constant release of 2 nm/ml for fetal Ch and 20 pm/ml for fetal ACh, 720 nm.Ch and 7.2 nm.ACh were released during that hour into the fetal circulation. One experiment gave substantially higher fetal ACh levels. These ranged in the order of 100-300 pm/ml. 75 Figure 14. Diagramatic representation of high voltage electrophoresis run showing separation of acetylcholine (ACh) and tetraethyl ammonium (TEA), choline (Ch) and tetramethyl ammonium (TMA) after one hour. See Materials and Methods for experimental conditions. 76 | CH O o O ACH O o O TEA ORIGIN 1 Figure 14 77 .maowuwvcoo Hmuooafiuoaxo you avenue: use mamauoumz mom .oaeamm HE A .mso mucomonemu uoaoo comm .ouowasm moaamfiumomzco zeloaxa vmofioucoo concomumm .:0Hwomuoa Honouraeo woo: woo m moan—6 oummowuma Houseman. ou mommoaou mmsHm> A.v mafiaoeoaxumom mom AOV mowaono .me mesmee 78 pm ACETYLCHOLINE 9, ml PERFUSATE C) CD F 1000 500 25 0 14 C) Q? P SiVSfHHSd IW/ (0) ENI‘IOHS W“ 15 20 10 TUBE NUMBER Figure 15 79 .mooauaoooo Housoaauoexo now moonuoz com masaumum: mom .oamamm as a .oso musomoumou uoeoa comm .ousmasm megawaumomhna Zeloaxa mosfimuooo ouomsmuom .sofimouuoa Honoucaao moo: moo m woes—6 oucmomuoa Houom on vommoaou mood; .A .v osaaosoahuoom use AOV 06:96 .ee «pause 80. pm ACETYLCHOLINEU/ ml PERFUSATE C3 CD CD 9 m 8 3 ~_ r’ I I l Ci ‘1 1O 1. <3 1. <3 4. 1O r 6 CD (3 I 1. l 1 l U) Vt 09 (M '- SlVSHdUBd lw/ (0)3Nl1OHO W" 15 20 10 TUBE NUMBER Figure 16. DISCUSSION A. Phospholipid Incorporation of 32Pi From.the results of this investigation, it appears as if the human term placenta cannot be added to the growing list of tissues which 4 respond to ACh addition by a change in the turnover of certain acidic phospholipids. In particular, these are phosphatidic acid (PA) and phosphatidyl inositol (PI). To our knowledge, these attempts were the first to try and establish this phenomenon, "the phospholipid effect", in zi552_using a physiologically normal tissue which lacks innervation yet contains such striking concentrations of ACh. Carbamylcholine has failed to produce a change in phospholipid turnover in neoplastic neuro- blastoma and glioma cell lines maintained in tissue culture (75). These cells, however, still have genetic characteristics similar to the inner- vated parent neural cell (76). Phosphatidic acid and PI contained 452 of the label incorporated into polar phospholipids, although they make up less than 52 of these phopholipids. It has been reported that in rat brain where these two substances are present at about 62 by weight, 992 of the label is in- corporated after a shorter incubation time (73). The probable reason for our lower percent incorporation was a longer incubation time which allowed more label to enter other compounds. I don't believe this affected our results because no phospholipids showed an increase. If 81 82 the cycling between PA and PI was increased initially, I would still expect to see some reflection of this. If initially the cycling was increased, but for some reason had slowed down, one might expect to see some of the other phospholipids reflect that increased incorporation of 321:1. Since the pool of these substances is quite small compared to some of the other phospholipids, the explanation for their intense labelling would be their rapid turnover. This turnover could be the result of two reaction sequences. The first would be the cycling of 1,2-diacylglycerol via PA and PI, shown on Figure 2, which involves two phosphorylation steps. The other would be the $3.2232 synthesis by phosphorylation of glycerol. Since treatment of placental tissue with cholinergic agonists did not change the fraction of 32P1 incorporated into PA and PI, the mechanism underlying the intense labelling was not explored any further since it probably was due to an unrelated metabolic process. Under the experimental conditions, the placental fragments would be expected to continue some metabolic processes. From.the failure of ACh to alter the turnover of placental polar phospholipids, it appears that the function(s) of ACh in this organ cannot be related to "the phospholipid effect" of ACh observed in tissues with cholinergic innervation and the speculations attached to this phenomenon in terms of membrane permeability. This experiment does not settle the question of "the phospholipid effect" in innervated tissue. It would suggest, however, that it is involved in synaptic transmission. This might not be in a causal role, but at least as an effect, because it appears that ACh functions differently in the human placenta than it does in neural tissue. One characteristic of this 83 difference is the lack of "the phospholipid effect" in the human term placenta (see Results section)(15). The inability of exogenous ACh to alter phospholipid metabolism, however, does not rule out that ACh has a function in the control of transport processes and membrane permeability of the placental membranes by a mechanism unrelated to their phospho- lipid turnover. B.l.'Membrane Potential Generation Based on one report in the literature with resepct to humans (28) and one on rabbits (31), an attempt was made to clarify the question of the existence of an electrical potential across human fetal membranes. we also wanted to see if ACh might have any effects on it. The rationale being that ACh might alter membrane permeability, in this case to ions, thus leading to physiological consequences. In this case, the generation of an electrical potential, or its alteration, was seen as a measurable parameter which would allow us to quantitate a cholinergic involvement in placental functioning. It was not possible to find any evidence for the generation of an electrical potential or any response to the addition of ACh. It is difficult to reconcile this with the one report in the literature. It is hard to draw direct analysis with the work done on the rabbit allantoic membrane (31). This membrane is not identical with the human fetal membranes that were examined. There also is a quantitative differ- ence between man and non-primate animals with respect to ChAc and ACh presence (7) . Based on this work, I do not believe the human term fetal membranes, amnion, chorion, and amniochorion are capable of producing a potential 84 difference. This might have been due to our experimental design, but it would be very difficult to further reduce the potential for trauma to the tissues. 24 2. Membrane permeability to Na From these experiments, it can be concluded that there is no active movement of sodium.ions across the examined human fetal membranes. As in the potential generation experiments, no sodium.movement occurred other than simple diffusion. Acetylcholine, at a final concentration of 1x10"4 M, did not have any effect on the movement of sodium. These findings are in opposition to one report in the liter- ature (28), as has already been mentioned. Our findings agree with one report (79) that states that sodium.movement is passive across the amnion and amniochorion. From this set of experiments, it can be concluded that even though the human fetal membranes contain ChAc (7), ACh does not play a role in permeability changes. It might, however, affect some other parameters. C. l. Cholinergic effects on amino acid uptake The experiments on the uptake of (140)AIB have shown that this nonmetabolized neutral amino acid was taken up against a concentration gradient. The linearity of this process with respect to time, the concentration ratios achieved, and the stimulating effect of tissue preincubation were in general agreement with the results of others (58,81). The results obtained with the tissue fragments indicated that manipulation of the functional state of the cholinergic system by drugs could alter the tissue accumulation of (14C)AIB. However, no unequivo- cal indication could be derived from the results as to whether cholinergic 85 manipulation stimulated or depressed amino acid accumulation. A pro- posed working model (5) considered both agonistic and antagonistic actions of ACh with respect to trophoblast permeability. This was depending on the amount of ACh release. Based on the effects of musca- rinic blocking drugs, in particular atropine sulfate which reduced the concentration ratios, it could be concluded that the action of ACh would enhance (140)AIB uptake. This, however, was not the case when ACh, muscarine and nicotine were added directly. On the other hand, paraoxon, generally thought to act indirect— ly by inhibition of acetylcholine esterase, also reduced the concentra- ting ability of the tissue even though only after 2-3 hours of exposure. A similar effect has been briefly reported (33) and was attributed to ACh accumulation as a consequence of.inhibition of the hydrolytic enzyme. The present results do not support that interpretation because physostigmine at lxlO-AM could be expected to cause as complete an inhibition of the esterase as lxlO-SM paraoxon. Physostigmine, however, had no effect on (l4C)AIB uptake. It has been reported that organophosphorus cholinesterase inhibitors, and paraoxon in particular, have a variety of biochemical effects which appeared to be unrelated to the blockade of ACh degrada- tion. Among those were direct effects on the cholinergic receptor (82). These effects included reversible inhibition of the receptor at high concentrations. This might explain its atropine like effects. Thus it appears conceivable that the effects of paraoxon were not due to ACh but more testing is necessary to separate its positive from.aegative actions on ACh and its receptor. 86 The reduction of (14C)AIB accumulation resulting from atropine, but not from d-tubocurarine, was caused only by concentrations which by criteria of drug action on innervated tissues must be considered ex- cessive. The effects were present in some experiments at 0.5 mM, but no longer at 0.2 mM. Nevertheless, it would be premature to reject the I idea of a muscarinic-like site in the placenta. A published report (10) has shown that atropine at 7.69 uM.depressed the release of ACh from floating villous tissue, an effect suggesting a muscarinic-type recogni- tion site. So while these experiments do not delineate a specific function for ACh in the human placenta, they strengthen the possibility for the presence of cholinergic recognition sites. This was tested for in the next set of experiments. 2. Examination of possiblegplacental cholinergic recognition sites The results of the attempts to attach drugs which are of proven value in tagging cholinergic recognition sites of either the muscarinic or nicotinic type, as differentiated in nervous tissue (62,63,65), were altogether inconclusive. (3H)ACh bound by what appeared to be unspecific adsorption to placental constituents while nicotine did not bind at all. Nicotine (57.9 mM), however, markedly stimulated the release of ACh from isolated villous tissue, while in higher concentra- tions (0.766 mM) it depressed ACh.release (10). Both circumstances suggest the presence of a nicotinic recognition site. However, in these experiments the use of (3H)nicotine concentrations as high as 10 uM still gave no indication of any binding whatsoever. The observations concerning the attachment of the more classical blocking drugs for muscarinic sites (133]QNB) and nicotine sites ([lzsljo-BT) were equally 87 questionable. With respect to the latter drug these results and conclu- 12511a-BT bound to placental consti- sions agree with Kau g£_al, (83). [ tuents but the binding could not be altered by the presence of an excess of unlabelled toxin making it unlikely that the binding was related to those cholinergic sites which have been described in brain tissue and in peripheral innervated structures (62,78). Although our results on the modulation of (14C)AIB uptake by atropine and more so the effects of that drug on ACh release (10) seemed to indicate a muscarinic type recognition site, the binding of (3H)QNB must be considered nonspecific. No drug which could reasonably be expected to interfere with (3H)QNB binding (78) had any effect. There are only a few published reports which deal with the presence of cholinergic recognition sites in tissues without innervation. One preparation was the chick amnion, a noninner- vated smooth muscle. An irreversible labelled muscarinic antagonist bound with the high affinity and specificity expected from a muscarinic type cholinergic receptor (35). The other system was sea urchin sperm cells where the experimental observations suggested a nicotinic type recognition site. This was very sensitive to stimulation by nicotine at low concentrations but inhibited by large concentrations. This was blocked by a-BT (85). The advantage of both models cited was that there existed a clearly defined physiological function which could be used as a reference. This was smooth muscle contraction or sperm motility. There is another non—innervated membrane, the rabbit allantois mentioned earlier, which responded to cholinergic drugs by changing its permeabi- lity as shown by electrical potential changes (31). 88 The results of this study allow a variety of interpretations. One might want to conclude that there was no cholinergic recognition sites in the human placenta. The pharmacological evidence stated above did not warrant that decision. The classical labelled antagonists could be unsuited for this noninnervated tissue because their chemical configura- tion did not conform to the structural prerequisites of the placental cholinergic recognition sites. Another interpretation could be that there were very few cholinergic sites in the placenta and that their affinity for the drug was extremely high. Under such conditions, which appeared unlikely because of the high drug concentrations required to change functional characteristics such as amino acid uptake and ACh release, the nM levels of drugs could be too high and lead to substan- tial nonspecific adsorption. This would lead to hiding the small amounts specifically bound. It was impractical to use lower drug concentrations because the available specific radioactivity was limiting. Alternative- ly, the postulated placental cholinergic sites could have low affinity for the drugs. This could also lead to significant nonspecific binding due to the concentrations needed thus making it impossible to distin- guish specific binding. Following the completion of this work, an unpublished study was found in which the saturable, reversible, high affinity binding of cholinergic drugs to human placental fractions was explored (34). Several of the drugs employed were identical with those used in this work. The conclusions drawn with respect to the presence of cholinergic recognition sites were comparable to that study. 89 D. cGMP Respgnse to ACh This set of experiments was undertaken to test if there might be an analogy between the human placenta and the neuronal response to ACh in terms of alteredchMP levels. When the results are viewed in toto, I believe it shows that there is no response to the addition of ACh, or the other cholinergic drugs tested. There are individual cases of statistical significance, and some cases that on appearance seem to be, but there are no trends. It is possible that there was a problem with our assay and the compound being measured was not cGMP. This was not the case based on the experiments discussed in Table 6. An experiment done by another member of our lab also lends support to the correctness of this assay technique. It was found that a claim in the literature (80), that ACh increased cGMP in human umbilical arteries, could be qualitatively reproduced. While these experiments only produced "negative data", they were still productive. As was stated in the Introduction, we were trying to determine if ACh has a role in human placental physiology. In order to do that, it is necessary to identify measurable parameters that would be involved with this ester's action. There is at the present time almost no published information that is useful. This set of experiments, as with all of than reported in this thesis, was done with the hope in mind of establishing a few parameters that could shed light on this perplex- ing problem. All of the experiments reported so far have not contributed to any significant breakthrough towards the solution of this question. They 90 have filled in a lot of gaps in the collective knowledge of placental functioning, however, and serve as a useful foundation for future studies. The following set of experiments, while only preliminary, provide the assay techniques that eventually will lead to meaningful answers. E. ACh Release from Perfused Placenta These experiments proceeded only through a preliminary stage due to the excessive work necessary to perfect the ACh analysis procedure. For reasons which still are not understood, seemingly straightforward extrac— tion and analysis procedures did not work. The best explanation avail- able is that one or more salts present in the perfusate buffer competed with substrates and/or inhibited the enzymes present. The final extrac- tion and analysis procedure takes one week to complete following a one hour perfusion. A spontaneous release of ACh from villous fragments of about 36 pm/gm tissue/min was reported (10). Based on an average placental weight of 500 gm and the weight of a cotyledon of 25 gm, the maternal ACh release shown in Figure 17 would equal about 168 pm/gm tissue/min. The fetal release of ACh would be about 5 pm/gm tissue/min. The higher maternal release value of this report is expected since cotyledon per- fusion would be more efficient. Two very important questions arise from.this work that have not yet been answered. Is the ACh that is released newly synthesized or is it the release of stored material? Also, why was there such a great disparity in ACh values for one of our experiments? Perhaps it was due to a physiological state that could be monitored in the future. This might provide an insight into placental ACh functioning. 91 This set of experiments has raised more questions than it has answered. It is, however, the best present method for analyzing possible ACh contributions to placental functioning. It (bilateral perfusion) will provide a direct test of placental permeability and therefore its influencing factors. SUMMARY In retrospect, questions might be raised concerning the choice of experiments. At the time of their conception, however, they appeared to be logical starting points for the analysis of acetylcholine function in the human placenta. The literature basis for this work is very minimal as illustrated in this thesis. No monitorable parameters existed at the inception of this work. Unfortunately, these studies did not lead to the identification of any. The time and effort was well spent, however, as a greater foundation now exists for future human placental study. While most of the results of this study were negative, they cannot be construed to mean that acetylcholine has no physiological role in human placental functioning. It only shows that the ones chosen were not directly applicable. The use of the bilaterally perfused placental preparation should go a long way in answering these questions. It provides a measurable parameter that is very close to the normal physio- logical process of transmembrane movement of nutrients. This more physiological type of experimentation is what is needed in the future. 92 BIBLIOGRAPHY 10. ll. BIBLIOGRAPHY Chang, C.H. and Gaddum, J.H. Choline esters of tissue extracts. J. Physiol., London, 12‘ 255-285, 1933. Fujiyama, T., Yamaguchi, R. and Noda, K. Nerve distribution in human fetal appendages particularly in the umbilical cord. Toh. J. Haupstein, P. Acetylcholine in der menschlichen placenta. Arch. fur Gynak. 151: 262-280, 1932. Heirman, P. L'acetylcholine placentaire. Arch. Int. de Physiol. _5_1_: 85-96, 1941. Harbison, R.D., Olubadewo, J., Dwivedi, C. and Sastry, B.V.R. In: "Basic and Therapeutic Aspects of Perinatal Pharmacology". P.L. Morselli, S. Garattini and F. Sereni, eds., pp. lO7-120, Raven Press, New York, 1975. Bull, G., Hebb, C. and Ratkovic, D. Choline acetylase in the human placenta at different stages of development. Nature 190: 1202, 1961. -Welsch, F. 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