PROLACTIN STIMULATION OF AMINO AGED-“C UMAKE mo mcoapoaAHON mm nmsm W M meson: CROP MUCOSA The“: 500 NM Dogma of DE. D. MICHIGAN STATE UNIVERSITY James A. Rillema 1968 . x ,. . I name _ we; ; LIBR.4"" M 6 Michigan Start 3 University [I 3 This is to certify that the thesis entitled PROLACTIN STIMULATION OF AMINO ACID-14C UPTAKE AND INCORPORATION INTO PROTEIN IN THE PIGEON CROP MUCOSA presented by James Alan Rillema has been accepted towards fulfillment of the requirements for M degree in My ‘T’flffiflw "/1 C/L/ Mailor professor Date Angust 7, 1968 0-169 ’~ -..... .. "’ rw-h—g—fl m1“ m ABSTRACT PROLACTIN STIMULATION OF AMINO ACID-14C UPTAKE AND INCORPORATION INTO PROTEIN IN THE PIGEON CROP MUCOSA By James A. Rillema Since the pigeon crop proliferates specifically in response to prolactin, it was intended to describe the regulatory action of exogenous prolactin on the pigeon crOp mucosa. Four to six—week old White King Squabs were ad- ministered prolactin subdermally over one side of the crop- sac; the other side was injected with a control solution. Following the hormone treatment, the uptakes into the epithelium were determined for the following radiolabeled 14C-amino acids, l4C-leucine, l4C-a-aminoisobutyric acid (AIB), l4C- 22N substances injected intravenously: hydrolysate sucrose, 3H-uridine, 3HOH, and a. A prolactin-enhanced l4C-amino acids began at nineteen uptake of hydrolysate hours following the injection. The prolactin-increased uptake of the nonmetabolizable amino acid, l4C-AIB, suggests that utilization of amino acids for incorporation into protein is not essential for an increased uptake. Prolactin also stimulated the uptake and incorporation of James A. Rillema l4C-leucine, but its enhanced uptake was less than the uptake of the hydrolysate mixture. Eg’yiyg prolactin-treated crop tissue was removed from the birds and the more superficial layers of the crOp were removed. The epithelium was then placed in a Lucite chamber and was bathed on both sides with bird Ringer solutions. The prolactin-treated tissue had a greater uptake of l4C-leucine and 3HOH added to the bathing solu- tion than the control "epithelium" preparation. The epithelium was therefore designated as the primary target for prolactin in the pigeon crOp. Enhanced protein synthesis in response to prolac- tin was demonstrated by isolating the protein from the free 14C-amino acids in prolactin-treated and control cr0p tissues and then measuring the activity of 14C in the TCA protein precipitate. The prolactin-enhanced protein synthesis and amino acid uptake (l4C-leucine) was inhibited by actinomycin D, thereby indicating that prolactin stimu- lates a DNA-RNA dependent protein synthetic mechanism. The fact that puromycin was unable to inhibit the uptake and incorporation into protein of l4C-leucine indicates that either the prolactin effect is puromycin insensitive, or the puromycin dose was too low to inhibit the prolactin reSponse, or prolactin counteracts the puromycin effect. James A. Rillema The uptake of 3HOH was increased at four hours and following fifteen hours of prolactin stimulation. These increases are probably related to metabolite movements: the four—hour increase is accompanied by enhanced uptakes of 22Na and 3H—uridine while the persistent hydration following fifteen hours is probably related to the increased amino acid and other metabolite uptakes. The l4C—sucrose space, "extracellular space," in the crop-sac was increased by prolactin administration, but this enlargement was not large enough to account for the total radioactivity of the accumulated amino acids and other metabolites. Electronmicrographs of the eighteen-hour prolactin- treated crop-sac show two regions of the epithelial layer to be enlarged: the stratum spinosum and the stratum basalae. Stimulated cell division is also indicated by the characteristic aggregation of the chromatin material in the nuclei of the prolactin-treated cells. PROLACTIN STIMULATION OF AMINO ACID-14C UPTAKE AND INCORPORATION INTO PROTEIN IN THE PIGEON CROP MUCOSA By fl James ATqRillema A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY_ Department of Physiology 1968 ACKNOWLEDGMENTS I would first of all like to thank Dr. William L. Frantz, Department of Physiology, for his guidance and interest in my development, both as an individual and for becoming a competent researcher in physiology. Apprecia- tion is also extended to the other guidance committee members, Dr. W. D. Collings, Department of Physiology, Dr. J. Meites, Department of Physiology, Dr. A. J. Morris, Department of Biochemistry, and Dr. E. P. Reineke, Depart- ment of Physiology. I am greatly indebted for their suggestions and revisions. For the use of the ultrasonic homogenizer, gratitude is expressed to Dr. J. R. Hoffert, Department of Physiology. I would also like to recoqnize the National Science Foundation for granting the funds designated for this work, and acknowledge the National Institutes of Health for pro— viding a traineeship.v ii TABLE OF CONTENTS ACKNOWLEDGMENTS o o o o o o o o o o o o I o o o o 0 LIST OF LIST OF Chapter I. II. III. IV. TABLES O O O O O O O O O O O O O O O O O FIGURES o o o o o o o o ' o o o o o o o o 0 INTRODUCTION 0 O O O O O O O O O O O O 0 THE LITERATURE SURVEY . . . . . .'. . . .' Prolactin-Stimulated Uptakes in the Cr0p . . . . . . . . . . . . Thermostability of Prolactin . . . . Antibiotics . . . . . . . .~. . . . . AIB . . . . . . . . . . . . Prolactin-Induced Structural Changes in the Pigeon CrOp . . . . . . . . MATERIALS AND METHODS o o o o o o o o o '0 The Experimental Animals . . . . . The In Vivo ExPerimental Protocol . . Sonicate 3V Vs. Intact Membranes . . . Prolactin Vs. Calf Serum . . .-. . . Growth Hormone Effects . . . . . . . In Vitro Uptakes in In Vivo Treated —Crop-Sac Epithelium . . . . . . . . Protein Synthesis Determination . . . Inhibition Studies . . . . . . . . . Electron Microsc0py . . .-. . . .~. . RES ULTS O O O O O O O I O O O O O O O O O 14 Hydrolysate C-Amino Acid Uptake . . 3IiOI-I Uptakes O O I O O I I O O O O O 14C-AIB Uptake o o o o o ‘0 o o o o o 3 . . 22 H-Ur1d1ne and Na+ Uptakes . . . . iii Page ii vi Us.) \Dmflh 10 12 12 12 16 17 18 19 20 21 22 23 23 28 33 33 Chapter VI REFERENCES . APPENDICES . 14 14 In Vitro Uptakes Amino Acid Availability and Incorporation The Method of Increased Protein Synthesis C-Sucrose Uptake . C— Leucine Uptake . into Protein . Electron Microscopy DISCUSSION iv Page 35 37 37 39 47 50 53 62 67 LIST OF TABLES Table Page 1. Radioactive nuclides used . . . . . . . . . . 13 2. Drugs and hormones used . . . . . . . . . . . l4 3. The effect of boiled prolactin on the uptakes of hydrolysate C—amino acids and HOH into the pigeon crop . . . . . . . . . . . 19 4. The effect of prolactin on the uptakes of hydrolysate l4C-amino acids and 14C- aminoisobutyric acid; the effect on the incorporation of hydrolysate l4C-amino into protein; and a comparison of sonicated vs. intact membranes of the pigeon crop . . 34 5. The effect of two to six hours of prolactin stimulation of the pigeon crop on the uptakes of 3H—uridine and2 Na . . . . . . . 36 6. The effect of prolactin on the 3HOH and C-sucrose spaces in the pigeon crop . . . 38 7. The effect of prolactin on the uptakes of 3HOH and l4C-leucine in vitro into the crop—sac epithelium . . . . . . . . . . . . 39 8. The effect of actinomycin D, prolactin, and actinomycin D with prolactin on the uptake 14 and incorporation of C—leucine and 3 HOH I I I I I I I I I I I I I I I I I I I 4 8 9. The effect of puromycin, prolactin, and puromycin with prolactin on the uptake and incorporation of l4C-leucine and HOH I I I I I I I I I I I I I I I I I I 0 4 9 Figure 1. LIST OF FIGURES Page T/C ratios for hydrolysate 14C-amino acids as a function of time after a single 25ug intradermal injection of prolactin at 0 hours . . . . . . . . . . . . . . . . 25 T/C ratios for hydrolysate 14C-amino acids in response to single, intradermal in- jections at 0 hours of prolactin in the amounts indicated on the curves . . . . . 27 T/C ratios for 3HOH as a function of time after a 25ug intradermal injection of prolactin at 0 hours . . . . . . . . . . . 30 T/C ratios for 3HOH in response to the amounts of injected prolactin indicated on the curves . . . . . . . . . . . . . . 32 The separation of unbound label recovered in the plasma vs. time following a single 5 uC/Kg injection at 0 hours . . . . . . . 42 14 The unbound hydrolysate C-amino acids and l4C-AIB recovered in crop membranes vs. time following a single injection IV of Sue/Kg of label I I I I I I I I I I I I I 45 Photomicrograph of untreated pigeon crop mucosal epithelium, x 4400 . . . . . . . . 52 Photomicrograph of eighteen-hour post pro— lactin-treated (25ug) pigeon crop mucosal epithelium, x 4400 . . . . . . . . . . . 52 Dual label, 3H and 14C, quench correction curves using an external standard . . . . 70 vi LIST OF APPENDICES Appendix I. Quench Correction in Dual Label Studies II. The Counting Solution . . . . . . . . . III. Composite Data of Radiolabeled Studies Page 68 o o I 72 o o o 73 CHAPTER I INTRODUCTION The homeostatic regulation of cells and cell organelles appears to be intimately related to hormones. And the mechanism of this relationship has been the sub- ject of much recent inquiry; it was thus shown that specific metabolites are accumulated and incorporated into cellular products in response to specific hormones. But similar responses have not as yet been clearly defined for metabolite movements regulated by prolactin. The production of crop "milk" and the simultaneous proliferation of the pigeon crop-sac are induced by the administration of exogenous prolactin alone. For studying the mechanism of action of prolactin, this organ therefore appears to be very useful. The enhanced uptake and incorporation of amino acids into protein and imbibition of water are integral events in the proliferative processes in many tissues; and the hormone control of some of these processes has been reported. It is the author's intent to relate pro- lactin stimulation of the pigeon crop to the time sequence of amino acid uptake and incorporation into protein; the 3 HOH uptake in response to prolactin was concurrently investigated. Subsequently, knowing the enhanced amino acid re- lationship to the crop-sac as regulated by prolactin, the mechanism of prolactin's effect was studied with the use of antibiotics and the nonmetabolizable amino acid, a- aminoisobutyric acid. The description of the prolactin- induced changes in the pigeon cr0p may have general application to the elucidation of the mechanism(s) of action of hormones. CHAPTER II THE LITERATURE SURVEY The crop-sacs of pigeons and doves (Order Columbiformes) produce a nutrient milky substance which they regurgitate and use to feed their progeny. This sub- stance was first called crop or pigeon "milk" by Hunter (1786, from Dumont 1965) and also by Bernard (1859, from Dumont 1965). An anterior pituitary hormone, prolactin, has a stimulatory effect on milk formation in these birds as well as in mammals. Davis (1939) found the crop milk to contain the following components: 33.8% fat, 58.6% protein, 4.6% ash, and 3.9% starch in the 28% dry matter. Therefore, it is the objective of this study to see how prolactin affects the enhanced protein formation in the pigeon crop. These results may then give further insight into the regulatory role of prolactin in the mammary gland and its tumors. The effect of anterior pituitary extracts on crop membrane proliferation was initially reported by Riddle and Braucher (1931), and prolactin was eventually isolated from pituitary extracts by Riddle gt 21. (1932-1933). Its molecular weight was found to be about 26,000 (Dixon and Li 1964). Others (Bassler and Forssmann 1964, Dumont 1965, Forssmann 1965, Meites and Nicoll 1966, and Weber 1962) have shown the specificity of prolactin for crop-sac epithelium and a consequent increase in mitosis (Leblond and Allen 1937 and Lahr and Riddle 1938). The crop-sac growth has also served as a basis for some commonly used prolactin bio-assays (Bates gt a1. 1963, Bergman gt il‘ 1940, Lyons 1937, Nicoll 1967, and Riddle EE.El° 1933). Prolactin-Stimulated Uptakes In the Crop McShan 22 El. (1950) commenced the time sequence study of some of the biochemical changes induced by exo- genous prolactin stimulation of the pigeon crOp. They found prolactin to cause a progressive increase of the succinate dehydrogenase activity which they took as an index of the metabolic activity. This increase proceeded linearly for four days at which time the activity level was four times that at zero time. Also their graph seems to indicate that the activity increased within the first four hours after the prolactin administration. Pentose nucleic acid, which is a measure of the ribonucleic acid (RNA) con- tent, was found to almost double between the first and second days of prolactin stimulation. Prior to one day, no notable increase was found. The desoxypentose nucleic acid (DNA) content and percent dry weight decreased for the first day following prolactin administration and then ||I|nlllrbfib pr, . progressively increased over the next forty—eight hours. The total crop-sac weight increased over the entire five- day period. Brown 32 a1. (1951) studied the uptake of 32 P into the crop as regulated by the daily intramuscular injection of prolactin. The dose-related uptake reached a maximum one hour following the radioactive nuclide injection on the fourth day; no further increase was observed. This probably means that the 32 P is no longer available after being in the plasma for more than one hour. They also found the prolactin-increased uptake was almost entirely in organic material, thereby indicating an increased in- corporation into DNA, RNA, and other phosphorous-containing organic compounds. By injecting prolactin daily for four days in localized subdermal injections over the crop-sac, Damm £5 31. (1961) essentially repeated the experiments of Brown 23 £1. Total phosphorus content was significantly higher as was 32F uptake, following a four-hour label time. The advantage of using the local injection technique is that variability among birds is eliminated from statistical consideration. Using a subdermal injection protocol for prolactin, five times in three days, Ben-David (1967) studied its regulatory effect on the uptake of 3H-methyl-thymidine in the cr0p epithelial cells. He reported a log-dose uptake of the label, which was in the birds between two and three hours. These data verify the increased DNA synthesis as reported by McShan st 21. (1950) and also substantiates the increased mitotic activity reported by Leblond and Allen (1937) and Lahr and Riddle (1938). Dumont in 1965 by means of electron microscopy reported an increased fat accumulation in the crop epith- elial cells twelve hours after prolactin treatment. Sub- sequent chemical analysis showed this fat to be primarily triglycerides. Also accompanying the enhanced accumula- tion of fat was an apparent increase in pinocytotic activity; this is one characteristic of the initial stages of lipid accumulation in cells. At twelve hours following the pro- lactin treatment, Dumont also demonstrated an increase in polysome formation; thus an increased protein synthesis is indicated. Sherry and Nicoll (1967) studied the relationship of prolactin stimulation to protein and RNA synthesis by using the antibiotics puromycin and actinomycin D. They found an inhibition of the crop-sac response to prolactin, thereby showing that the increases in RNA (inhibited by actinomycin D) and protein (inhibited by puromycin) are integral parts of the prolactin-stimulated proliferation. These antibiotics, however, were only able to inhibit partially (20% to 30%) the response to locally injected prolactin. The inhibitions reported by these investigators were at forty—eight hours for puromycin (200ug) and at twenty-four hours for the actinomycin D (0.5ug). The prolactin-induced increase of RNA and protein synthesis are therefore present by at least the times indicated. Sherry and Nicoll (1967) also reported a crop-sac response to intradermal injections of RNA extracted from the crop epithelial cells of prolactin—treated birds. Although a proliferation of the crop-sac was evident, this only indicates, but does not prove, that the prolactin- induced proliferation is dependent upon the synthesis of new RNA, especially since actinomycin D was able to inhibit only 20% to 30% of the prolactin-induced crop proliferation. Thermostability 9f Prolactin In 1933 Riddle 93 31. first reported the stability of prolactin boiled for twenty minutes at pH 7.5 to 8.5, but they gave no data to support their conclusions. A one- hour treatment under the same conditions again showed prolactin to be thermostable (McShan and French 1937). Geschwind and Li (1955) reported that growth hormone boiled for fifteen minutes at a neutral pH had no stimula- tory effect on tibial epiphysis growth in the rat. Whereas prolactin, similarly treated, had the same effect as unboiled prolactin on tibial epiphysial growth and also had its normal crop-sac stimulatory activity. Later Kostyo and Schmidt (1962) showed that prolactin boiled for twenty minutes at pH 8 maintained its crop-sac stimulatory activity while its effect on the AIB uptake of the rat diaphragm decreased. The authors there- fore indicated that growth hormone, which is‘destroyed by the boiling procedure, probably contaminated the prolactin and was responsible for the enhanced amino acid uptake. Hjalmarson and Ahren (1967) found that prolactin and growth hormone, boiled for fifteen minutes and administered in 2132, did not inhibit the in vitro growth hormone—stimulated AIB uptake in the rat diaphragm, whereas the non—heat— treated hormones did inhibit this response. But the heat- treated prolactin retained its ability to stimulate the mammary glands. Here again growth-hormone contamination of prolactin was probably responsible for the non-boiled- prolactin response. The thermostability of prolactin boiled for fifteen to twenty minutes at pH 8 is therefore clearly suggested by the available literature. Positive proof, however, that all the effects of prolactin remain following the heat treatment is lacking. That boiled growth hormone has less activity is well substantiated, but further experiments are necessary to verify the complete inactivation of growth hormone by the heat treatment. Antibiotics Actinomycin D and puromycin have been used rather extensively for showing how certain biological changes are effected. Actinomycin D apparently inhibits the tran- scription process (Hawamata and Imanishi 1960, Reich gt gt. 1961, and Rounds gt gt. 1960) by inhibiting RNA polymerase activity and forming a complex with DNA (Reich 1963). Puromycin appears to selectively prevent the amino acid transfer from t-RNA to protein, thereby preventing trans- lation (Nathans and Lipmann 1961, Rabinovitz and Fisher 1962, and Yarmolinski and De La Haba 1959) for protein formation. AIB Extensive use of AIB as a substitute for metaboliz- able amino acids has been made. It is actively transported across the human erythrocyte by the same processes as the neutral amino acids (Winter and Christensen 1963). Growth hormone increases the accumulation of AIB in the rat diaphragm cells (Kostyo gt gt. 1962) in a manner similar to an enhanced accumulation of the metabolizable amino acids. Other hormones, including thyroxine on embryonic rat bone (Adamson and Ingbar 1967), epinephrine on the rat uterus (Noall gt gt. 1957), estrogen on the rat uterus and rat excretion (Noall gt gt. 1957 and Riggs and Walker 1963), insulin on rat liver (Chambers gt gt. 1965), TSH on bovine 10 thyroid slices (Debons and Pittman 1966), and hydrocortisone on rat liver (Chambers et a1. 1965) also increase AIB accumulation in their respective target tissues. Prolactin-Induced Structural Changes tg the Pigeon Crop In 1963 Forssmann by means of electron microscopy reported increased folding, cell number, and cell volume in response to prolactin. Also, fat vacuole content of the crop cells was enhanced as were the numbers of mito— chondria, ergastoplasm membranes, ribosomes, and desmasome openings. Bassler and Forssmann (1964) reported similar findings, but they did not look at the initial twenty-four hours following prolactin administration. Folds of the epithelium into the lamina propria were reported by Masahito and Fujii (1965 from Dumont 1965) twelve hours after the prolactin treatment. These folds increase with time following the hormone injection. Dumont in 1965 took electron micrographs of twelve- hour, two-day, and four-day prolactin treated crop tissues. The stratum spinosum contained more lipid dropletts and had an increased pinocytotic activity following a twelve- hour prolactin treatment; polysome formation was also considerably enhanced. Intracellular cannals enlarged two days after the injection of prolactin, while the lamina propria contained many more vesicles. Also, after two days, the stratum basalae was enlarged. The stratum ll spinosum was shown to triple in thickness four days following the prolactin treatment. Dumont also reports an increased vascularity of the crop in response to pro- lactin, but he does not report when this process begins. Sherry and Nicoll (1967) show photographs of cr0p tissues treated with RNA extracts from other cr0p tissues. The proliferation of the RNA-treated tissues is obvious, but the relation of prolactin to this RNA stimulation is not clear. ——-.— ”'1“! CHAPTER III MATERIALS AND METHODS The Experimental Animals Four to six-week old White King Squabs (Cascade Squab Farm, Grand Rapids, Michigan) of both sexes were maintained on a normal diet, at room temperature (27°C), and were exposed to fourteen hours of light daily. The birds were maintained under these conditions three to ten days prior to experimentation; food and water were avail- able gg libitum for the duration of this "equilibration" period. The t2 Vivo Experimental Protocol Twenty-four hours prior to experimentation, the feathers of the squabs were removed from the entire crop- sac surface and from the axilla. Following this prepara- tion the birds received intradermally 0.1 ml of a NIH prolactin solution (Table 2), thereby forming a bleb over One lateral aspect of the crop-sac. The other half of the crop-sac received an equal volume of bird Ringer solution (Table 2), distilled water, or fetal calf serum (Table 2). The prolactin used was mixed in distilled water and kept frozen until injected into the birds. Since the author 12 13 Table 1 Radioactive Nuclides Used Specific Radioactive Nuclide Activity 3 HOH ---- 14 . C-protein 172 uC/mM hydrolysate amino acids 14 . C-leuc1ne 165 mC/mM 3H-uridine 5.0 C/mM 22Na 1.13 mC/mM 14 C-sucrose 480 mC/mM 14C-a- 10 mC/mM aminoisobutyric acid Lot Number 6606 6701 6702 22 19 103 6701 6701-P Company New England Nuclear Schwartz Bioresearch Inc. Nuclear Chicago Nuclear Chicago Abbott Laboratories Schwartz Bioresearch Inc. Schwartz Bioresearch Inc. 14 Table 2 Drugs and Hormones Used Substance Source Lot # or Activity Prolactin NIH Endocrin. Research Section 12.8 IU/mg PB-l* Prolactin NIH Endocrin. Research Section 19.6 IU/mg PB-2* Actinomycin Merck, Sharp, and Dohme L554651—0-10 D* Research Laboratories Puromycin Nutritional Biochemicals Corp. Control #4695 Fetal Calf Grand Island Biological Control #419088 Serum Inc. Bird Ringer (Frantz and Rose 1968) Solution * - Donated by the above named institutions found decreased activity of the hormone when the prolactin was in solution and kept frozen for longer than two weeks, prolactin solutions kept longer than this time were dis- carded. Prolactin activity also appeared to decrease more rapidly when the hormone was frozen in bird Ringer solu- tion, probably due to the salt concentration (Riddle gt gt. 1933). In preliminary experiments, it was ascertained that the effect on the crop epithelium of a single, intra— dermal injection of 25 ug of PB-2 prolactin remained unilateral. At specified time intervals subsequent to the hormone treatment, 5uC/Kg of each of the following 15 radio-labeled substances in combinations of two were 22Na+ 3H-uridine, tritiated 14 injected into the wing vein: 14C-amino acids, C-a-amino- C—leucine, and l4C-sucrose. (See Table water, protein hydrolysate isobutyric acid, 14 l for specific activities and sources of the radioactive nuclides.) At predetermined times, from one to six hours after the label administration, the birds were killed by cervical dislocation, and two to three cm2 discs of the prolactin and control-treated areas of the crop were excised, weighed, in most cases sonically homogenized, and counted. Simultaneously from each bird, heart-puncture blood samples were drawn, centrifuged, and a 100u1 aliquot of the plasma was transferred into the counting medium. The radioactivity within all the samples was determined in a modified Bray's (Bray 1960) dioxane scintillation solu— tion with a Nuclear Chicago Model 6860 liquid scintillation counter. Background counts were automatically subtracted, and quench correction was done by use of an external 133Ba standard; this technique allows for simple disintegration per minute determinations from dual labeled samples (see Appendices I and II for quench correction counting proce- dures). The data from membrane counts was expressed as a ratio of treatment over control, or T/C, where T represents the disintegrations per minute per milligram (DPM/mg) for the prolactin-treated tissues and C, the DPM/mg of the 16 control-injected counterpart. The use of T/C ratios, rather than gross counts of treated and control tissues, eliminates statistical variability among birds. The statistical test for the T/C ratios being significantly greater than 1.0 was the student t-test (Ostile 1963). When comparing the uptakes of the labeled amino acids (hydrolysate 14C-amino acids, l4C-AIB, and 14C-leucine), it was of concern that the specific activities of the various labeled substances (Table l) differed markedly. But with respect to the endogenous amino acid concentration in the plasma, which is about 3.0-7.0mM (Spector 1956), their circulating specific activities were nearly identical, gg. 1.666 x 10-9C/mM. Sonicated 2g. Intact Membranes In some experiments, counts were determined and T/C ratios were calculated from whole membranes placed in the scintillation medium. And in order to evaluate the validity of using this procedure, a series of membrane pairs was ultrasonically homogenized (Heat Systems Co., Sonifier Model W-185-c) for five minutes at 300 watt—minutes and then counts were determined in the manner described above. Although the total counts were greater, the re- 14 . . C-amino ac1ds were sulting T/C ratios for hydrolysate not different from those of the intact membrane samples (Table 4). In all cases, a twenty-four hour prolactin l7 pretreatment and four-hour label time were used with 5uC/Kg of each labeled substance injected. In homogenized samples about ten times more DPM/mg were counted for the l4C-labeled protein hydrolysate, and three times more were counted for the 3HOH. Counts within the prolactin-treated and control tissues increased proportionately with the result that the T/C ratios between intact and sonicated tissues were not different. All tissues except those from the dual labeled studies of 3HOH and hydrolysate l4C-amino acid uptakes were sonically homogenized prior to activity determinations. Prolactin 2g. Calf Serum Since prolactin is a proteinaceous compound, it is germaine to question whether a nonspecific protein stimula- tion of metabolite uptake in the pigeon crop would occur. Lahr gt gt. (1943) demonstrated a crop-sac proliferation with oils, bile, and many other substances. To establish its specificity, therefore, prolactin was administered as previously described, and fetal calf serum (700ug/ml) was used in the control injection. Its protein content, molecule per molecule, was the same as that of the injected prolactin. Calf serum was used for a control in all exper— iments except the dual label studies where 3HOH and hydro- lysate 14 C-amino acids were injected. Therefore, the various comparisons using the same time sequence for in— jections and sampling establish the specificity of prolactin 18 for causing the enhanced uptake and other changes which are reported (3HOH ratios in Figure 4 and Table 6). Growth Hormone Effects The amount of growth hormone present in NIH PB—2 prolactin is reported by NIH to be less than one percent. But, since growth hormone is able to cause an increased amino acid accumulation in other tissues, (Kostyo and Schmidt 1962), it was decided to test whether growth hormone is also a causative agent in the various enhanced uptakes observed with the prolactin solution. It is fairly well established, but not conclusively, that boil- ing at pH 8 for twenty minutes destroys growth hormone activity whereas prolactin retains its activity. Using a twenty-four hour prolactin treatment and a three-hour label l4C-amino acid and 5uC/Kg 3HOH), time (5uC/Kg hydrolysate a comparison was made between a 25 ug normal PB-2 prolactin injection and a 25 ug boiled PB-2 dose. The results (Table 3) show no difference between the T/C ratios of BHOH and the hydrolysate 14 C-amino acids of the two groups. It is concluded that there is no effective activity of growth hormone in NIH PB-2 prolactin. And also the stability of prolactin under the conditions listed above is further verified. _«I 19 Table 3.--The effect of boiled prolactin on the uptakes of hydrolysate C-amino acids and 3HOH into the pigeon crOp. PROLACT IN NORMAL BOILED l4c Amino Acid T/C 1.23:0.10 (9) 1.28:0.07 (10) 3HOH T/C 1.2610.06 (19) 1.35:0.13 (10) Mean i Std. Error (N) tg Vitro Uptakes tg tg Vivo Treated Crgp-Sac Epithelium For $2.21EEQ studies the bird's feathers were plucked twenty-four hours prior to a 25 ug intradermal injection of PB-2 prolactin over one side of the crop-sac; the control side received an equimolar injection of fetal calf serum protein. Twenty-four hours later, the hormone and control-treated membrane portions were excised, and the epithelia were isolated by teasing away with a forceps the more lateral layers of the crop. The membranes were then put between identical halves of Lucite bathing chambers. The tissue preparation and modified Ussing bathing appara- tus are described by Frantz and Rose (1968) and Rose (1967). Subsequent to a one-half to two-hour perfusion time for equilibration, where the membranes had bird Ringer solu— tions on both sides with constant stirring, 1.0 uC of 14C— leucine and 1.0 uC of tritiated water were placed in the serosal bathing fluid. A 100 ul sample of the "hot" 20 bathing fluid was taken and put in the scintillation counting medium. After a one or ten-minute label time, the fluid was removed from the bathing chambers, and the membranes were thoroughly rinsed once with cold bird Ringer solution. Then the bathed portion of the crop tissue was removed from the Lucite blocks and cooled immediately to st0p metabolic activity. The tissues were then weighed, placed in 1.0 ml water, sonically homogen- ized, and counted in the liquid scintillation fluid. Protein Synthesis Determination The procedures involved in the demonstration of an enhanced protein synthesis in response to prolactin are described primarily in the results section, thereby eliminating some unnecessary repetition. Briefly, the proteins from prolactin and control—treated cr0p tissues, sonically homogenized, obtained from birds injected with hydrolysate l4C-amino acids, were precipitated with 10% TCA and the activity of the protein then determined. The activities in the prolactin-treated and control tissues were then compared. The effect of t-RNA binding of amino acids on the protein precipitate counts was eliminated by washing the precipitates with 1N NaOH for less than two minutes. Then the protein was reprecipitated with 10% TCA and washed once more with the TCA. After the protein was again isolated by centrifugation, the activity in the 21 protein precipitate was determined and compared to that in the tissue homogenate. Inhibition Studies The manner by which prolactin exerts its influence was examined with the use of two antibiotics, actinomycin D and puromycin (Table 2). Puromycin (200 ug) was injected intradermally over one lateral aspect of the crop—sac; the other surface received an equal volume of bird Ringer solu— tion, the same medium used to mix the antibiotics. One hour later, one-half of these birds received 0.1 ml PB-l prolactin (0.5 IU) into the bleb where the puromycin had been injected; the control side received simultaneously a fetal calf serum injection. Seven other birds received prolactin and calf serum only, i.e. they received no anti— biotics. A protocol similar to that for the puromycin experiments was used for actinomycin D except that the dose was 0.5 ug. Twenty-four hours after the antibiotic or hormone treatments, the birds received intravenously 3HOH and 5 uC/Kg l4C-leucine. Three hours later 5 uC/Kg the treated and control tissues were excised, halved, weighed, and sonicated. One of the sonicated halves was then used for a total count determination by liquid scintillation counting; the other half was used for deter- mining the counts in the protein fraction. The procedure for the protein isolation and 14C counting was as previously described. 22 Both the puromycin and actinomycin D solutions were premixed in bird Ringer solution and kept frozen until used. Both were used for experimentation within a week, thus minimizing the loss of activity of these antibiotics. Electron Microscopy Eighteen-hour prolactin (25 ug of PB-2) and calf- serum-treated membranes were prepared for electron micro- scopy following an t2 ztzg hormone and control treatment. The tissues were excised and fixed in glutaraldeheyde buffered to pH 7.5 with phosphate. Then two hours later the tissues were post—osmicated for ninety minutes in phosphate-buffered osmiun tetroxide. As outlined by Dumont (1965), the tissues were further prepared by Mr. Gordon Spink of the Biological Research Center, Michigan State University. CHAPTER IV RESULTS 14 Hydrolysate C-Amino Acid Uptakes The uptake of hydrolysate l4 C—amino acids into the piegon crop epithelium in response to a 25 ug injection of prolactin is plotted in Figure 1. Zero time represents the beginning of a single PB-2 prolactin treatment, and the arrows on the baseline (T/C = 1.0) represent the sub— sequent intravenous radiolabel injections. The data points are mean T/C values (N 2 5); the vertical bars are the standard errors of the means. Hourly ratios for the hydroly- sate l4C-amino acids show an enhanced uptake beginning at nineteen hours after the prolactin treatment. The dotted lines represent best fit curves (visual) for average data points corresponding to separate isotope injections; the solid line represents a composite curve for the significant ratios up to thirty hours. Following a twenty-four hour PB-2 prolactin treat— ment, a dose-response relationship (Figure 2) of the hydrolysate 14C—amino acid T/C ratios is evident twenty- eight to thirty hours after the prolactin was injected into the birds. The plateau in the 25 ug curve can be 23 24 FIGURE 1 l4 . . . C-amino ac1ds as a function T/C ratios for hydrolysate of time after a single 25 ug intradermal injection of pro- lactin at 0 hour. Each arrow represents the injection of 5 uC/kg (173 uC/mMole) of amino acids. Each open circle, which corresponds to the immediately preceding arrow, is the average T/C t SE (N15) which is significantly greater than 1.0 (P<0.05). The solid circles are insignificant ones. The dotted lines are best—fit curves for the mean values derived from the 18 and 24 hour label injections. The solid line is the best-fit curve for the maximal up- take ratios of the two individual curves. 3‘va 20:09.2. z_._.o<._omm mm...m< mmDOI onmuomcmmmom 0.0. .v. N_o_ m w v N O 1 _ q q — q _ d _ 1 Ji _ d u I U‘ *HH‘. 1‘. ... .‘w‘. .1: £1.11... s \ IN.— I?.. I0; IQ.— IO.N mo_o< OZ_2< 0! o\.r I N.N 26 FIGURE 2 T/C ratios for hydrolysate l4 C-amino acids (filled circles, mean t SE) in response to single, intradermal injections at 0 hour of prolactin in the amounts indicated on the curves. For the control ratios (open circles) bird Ringer solutions were injected at 0 hour over both halves of the crop. Into all the pigeons 5 uC/kg (173 uC/mMole) of hydrolysate amino acids were injected iv at 24 hours; tissue samples were taken at intervals for six hours. Each point represents an average T/C t SE (N Z 5). 27 20:09.2. z_._.o<._omn_ much; mKDOI on mu 3 5 mu 3 LT I I T o¥\u¢\o W )H\ Sing”. m o\lflW\ HI‘ 8.} <3. H - H HI 8. \u.\o.n ~ 8.3 02.3 -o: o\» I? um. 10. 10.. IN.— :¢.. 1 0.. r m.— l QN 28 attributed to the limiting amount of labeled amino acids available from the plasma. 3HOH Uptakes Figure 3 shows the uptake of 3HOH in the pigeon crOp epithelium in response to a 25 ug injection of pro- lactin. The ratios are calculated and plotted in a manner l4C-amino acids similar to those of the hydrolysate (Figure 1). Between zero and fifteen hours following the prolactin administration no ratios were significantly greater than 1.0 except for the four-hour response. Ratios obtained from a repetition of the eXperiment covering the initial six hours, in which both prolactin and 3HOH were given at zero time and tissue samples obtained from one to six hours later, were not different from the original data; i.e. in only the four-hour samples were the T/C ratios significantly greater than 1.0. Following a fifteen-hour prolactin treatment, the T/C ratios for 3HOH are signifi- cantly greater than 1.0 for at least up to thirty hours. The ratios also appear to increase progressively for the duration of the fifteen to thirty-hour time increment. Following a twenty-four hour prolactin treatment, a dose-response relationship of 3HOH uptake is evident from twenty-eight to thirty hours after the label administration (Figure 4). The rate at which the T/C ratios fall toward 1.0 in the 1.25 and 12.5 ug curves appears to be a function 29 FIGURE 3 T/C ratios for 3HOH as a function of time after a 25 ug intradermal injection of prolactin at 0 hour. Each arrow represents an iv injection of 5 uC/kg of 3HOH. Each open circle, which corresponds to the immediately preceeding arrow, is the average T/C 1 SE (N Z 5) which is signifi- cantly greater than 1.0 (P<0.05). The filled circles are for the T/C ratios which are not significantly different from 1.0. C = 77.7 i 6.4 DPM/mg, N = 164. I383 zo_5m..z_ z_._.o<._oE Eta $50: on mm mm ¢~ Nu cu m. m. S a. o. o m l. N 0 IT _ _ _ _ _ _ _ _ M _ _ _ . _ \F 4 4 - w . LA » A? a o_ 0 WW m W I ~._ 3 It. -9. I Q. :2...» ob. 1 o.~ {>53 >51) m.*rl!""" 31 FIGURE 4 T/C ratios for 3HOH (filled circles, mean 1 SE, N 3-5) in response to the amounts of prolactin indicated on the curves. A single 5 uC/kg dose of 3 HOH was injected at 24 hours after the single, intradermal injection of prolactin. For the control ratios (open circles) bird Ringer solution was injected intradermally at 0 hour over both halves of the crop, then 5 uC/kg of 3HOH was given iv at 24 hours. c = 77.7 1 6.4 DPM/mg, N = 164. 20:09.2. Z_.ro pmumoHGOm mo camflummaoo m can «samuonm ousfl mpwom osflsmuova mummMHoupmn man so uommmm mzu Inflow UHHNDSQOmHocHEmloIU¢H cam mcflom ocflEMIU¢H can mcflom ocHEMIOvH mummhaouomn mo mwxmums msn so quomaonm mo pommmm mnBI|.v magma 35 can be seen in Table 5, the T/C ratios for both isotopes were significantly greater than 1.0 only at four hours following the prolactin treatment. The interdependence of the increased 3HOH, 22Na+, and 3H-uridine uptakes cannot be explained from this data. It is also pertinent to note here that T/C ratios (Figure 1) for the hydrolysate l4C— amino acids, simultaneously injected, were insignificant at the four-hour time following the prolactin stimulation. l4C—Sucrose Uptake In order to evaluate the effect of prolactin on extracellular space, the distribution of l4C-sucrose was determined twenty-eight hours following a PB-l prolactin (0.5 IU) treatment. 14C-sucrose (5 uC/Kg) and 3HOH (5 uC/Kg) were injected four hours prior to sampling; the 3HOH ratios thus served for comparative purposes with pre— viously obtained ratios. As can be seen in Table 6, the T/C ratios for l4C-sucrose and 3 HOH are both significantly greater than 1.0. And the population of 3HOH T/C ratios was not different from that of previously obtained ratios using a similar treatment (Figure 4). An enlarged l4C- sucrose space, "extracellular space," (Fenstermacher and Bartlett 1967) in response to prolactin is thereby indicated by these data. 36 mH.HNm.¢ N0.Hmm. on.who.mH .m.mHGmmZ mo.vm« Ams\zmave m u z ma.wam.a mm.fiom.¢ ¢~.qu.e 5N.Hmm.v mem o so.Hmo.H «mo.H-.H ao.nmm. mo.nmo.a mz- o\e mm.HH~.mH HR.HH¢.oN am.fim.ma mm.Hm.oNe wanna“: mm o mo.flmo.a OH.H>N.H so.fimo.a mo.Hmo.H mcfiwflno mm o\e omz mm m N Amusomv mzHe cam wcflcfluslm mo wmxmvms map so mono m sommflm on» no coaumaseflum GHDUMHOHQ mo mason xflm Op o3u mo buwmmm 0381:.m OHQMB 37 Table 6.--The effect of prolactin on the 3HOH and 14C- sucrose spaces in the pigeon crop. RADIOACTIVE NUCLI DE 3H0H l4c SUCROSE T/C 1.21i.lO* (5) 1.13:.04** (5) C = 32.5:2.7 DPM/mg 2.77:3.6 DPM/mg Mean S.E. *P<.05 **P<.01 14 C-Leucine Uptake 14 The t2 vivo uptake of C-leucine, which is one of 14 the constituents of the C-amino acid hydrolysate mixture, provided a basis for comparison with tg vitro labeled mem- branes. Using a twenty-four hour, PB-l prolactin (0.5 IU) 14 treatment, the birds received 5 uC/Kg C-leucine and 3 5 uC/Kg HOH; the samples were taken four hours later. 14 The T/C ratios for the C-leucine and 3HOH are both greater than 1.0 (Tables 8 and 9). The ratios for the tritiated water being in the same statistical pOpulation 14 as those obtained when hydrolysate C-amino acids were used indicate constant experimental conditions. The ratio 14 for the C—leucine (1.13) was greater than 1.0 but less 14C-amino acids‘ than those obtained for the hydrolysate (1.41) (Figures 1 and 2). A preferential uptake of other amino acids in the hydrolysate l4C-amino acid mixture 38 also appears evident since the average increased uptake of the hydrolysate mixture was 41%. tn Vitro Uptakes The demonstration of an enhanced uptake tg_ytttg of 3HOH and l4C-leucine in prolactin-stimulated crop tissues was attempted for two reasons. First, to show that endogenous factors are not immediately essential for the enhanced accumulation of the above two substances, once prolactin has affected the crOp tissue. And second, to show that the prolactin-enhanced accumulating capacity for amino acids is in the crOp epithelium and not in the muscle or other tissue. The crop-sacs were 25 ug PB—2 prolactin-treated for twenty-four hours; the control side received an equimolar concentration of fetal calf serum. The membranes were placed in identical bathing chambers and the isot0pes(3HOH and14C—1eucine) placed in the Ringer solution bathing the serosal surface of the crops. Then the tracer-eXposed area of the crOp was excised, blotted, weighed, and sonicated, and the radioactivity was determined. Following a tenwminute radioactive nuclide exposure .£E.X£E£21 no difference in counts was evident in the pro- lactin and calfwserum-treated tissues. But using a one- minute exposure time, T/C ratios were significantly greater l4 3 than 1.0 for C-leucine (p<.10) and HOH (p<.05) (Table 7). This demonstrates that the pigeon-crop mucosal epithelium, 39 stimulated tg ytyg, is able to accumulate amino acids and 3HOH tg ztttg in the absence of submucosal layers. But this does not mean that prolactin has no effect on the tissues which were removed from the bathed membrane portion. Table 7.-—The effect of prolactin on the uptakes of 3HOH and 14C-leucine tg vitro into the crop-sac epithelium. RADIOACTIVE NUCLIDE Eggg ggtgg iggg l4c LEUCINE 1 min. T/C 2.13:.27** (7) 1.75:.38* (7) 1 min. gfifigfié$9§5§$9§i. 0.474:.057 (7) 0.6121.090 (7) 10 min. T/C 0.93:.37 (7) 0.97:.08 (7) . C DPM/mg. memb. 10 m1n. DPM/mg. ser. fl. 2.3661.559 (7) 2.714i.354 (7) MeaniS.E. *P<.10 **P<.01 Amino Acid Availability and Incorporation Into Protein To better understand the manner in which labeled substances are available from the blood, a series of plasma samples from the radiolabeled birds were counted, centri- fuged, the dioxane supernatants removed, and both fractions were counted. The counts remaining in the supernatant pro- vided an estimate of the radiolabeled fraction that was not bound to plasma proteins. As determined by this dioxane 40 partitioning, the fraction of unbound l4C-amino acids in the plasma decreased exponentially in four hours to about 17% (lowest curve of Figure 5, filled circles). A plot of the accumulated count in the precipitated plasma protein produces a reciprocal curve which when added to the curve for counts in the supernatant totals 100% of the initial counts. To determine the affinity of nonpeptide-linked amino acids for the plasma precipitates, the dioxane- separated precipitates were centrifuged and shaken in 0.5 ml of a 10% TCA solution for one hour. These TCA precipi- tates were then washed twice with water and counted in fresh scintillation fluid. Washing with TCA increases the yield of the protein fraction by about 7% (open circles, Figure 5). The effectiveness of TCA in releasing absorbed amino acids and 3HOH in the absence of protein synthesis (Figure 5, zero hours) was demonstrated by mixing 0.25 uC/ml (equivalent to the 5 uC/Kg tg gtzg) of 3HOH and 14 14 either hydrolysate C-amino acids or C-AIB with pigeon plasma in a test tube for one hour; the plasma proteins of the separated samples were washed with TCA in the above manner and counted. After the TCA wash, 92.0 i 1.3% (N = of both the l4C-amino acids and 14C-AIB, and 96.0 i 1.3% (N = 10) of the 3HOH introduced tg vitro was released from the plasma protein fractions. Therefore, the fraction of 5) 41 FIGURE 5 The separation of unbound label recovered in the plasma vs. time following a single 5 uC/kg injection iv at 0 hr. (mean percent of the total count 1 SE, N 3 5). Hydrolysate 14C-amino acids are represented by circles, l4C-AIB by triangles, and 3HOH by squares. 3113 I ""1 31»’::» l by 42 IOO I4 % UNBOUND IN PLASMA 90 _ c AIB if; i J3HOH ao- - If 1I4 -, .. c AIB O L .. 3H0H T I +4 50- 404 § 30- ' I o l4c 20- AMINO ACIDS "c mmo u. moxms SEPARATED ACIDS 90A TCA WASHED l0 1 l l 1 l I o l 2 3 4 5 6 HOURS AFTER ”c AMINO ACID AII03II0II INJECTION 43 l4C-amino acid that remained in the plasma protein after the TCA wash (open circles, Figure 5), minus the 8% not removed at zero time, is inferred to be peptide-linked; this is approximately 60%. After a two—hour equilibration time tg vivo, 69% of the plasma 3HOH (second curve of 14 Figure 5, filled squares) and C-AIB (filled triangle) separated with the dioxane fraction. The TCA wash (open 14C-AIB of Figure 5) provides squares 3HOH, Open triangle a 20% increase in both unbound water and l4C-AIB; the re- maining 10% presumably represents label still bound to plasma protein. The determination of amino acid and water distri- bution in the two-phase system of the plasma provides a basis for interpreting their distribution in the more com— plex structure of the mucosal epithelium. Data for whole membrane samples, treated in a manner similar to those of the plasma, are given in Figure 6. The amount of hydroly- 14 sate C-amino acids unbound in the dioxane-counting medium when the pigeon crop membrane was taken three hours after labeling had decreased exponentially to about 25% of the total count. When the membranes were washed with the TCA solution before they were suSpended in the dioxane solu— tion, the quantity of the unbound hydrolysate 14C-amino acids was 43%. When the membranes were sonically homogen— ized, then washed with 10% TCA and 1N NaOH solutions, a 1 14 Similar proportion of the tota C counts was recovered 1.: L; 44 FIGURE 6 The unbound hydrolysate 14C-amino acids and 14C-AIB recovered in crOp membrane vs. time following a single injection iv of 5 uC/kg of label. Each data point is the mean percent of the total counts + SE, N Z 5. % UNBOUND IN MEMBRANES I00 90 80 70— 60— 30— 20— IO N-s We AIB (TCA WASH,SONICATED) I'4C AIB (TCA WASH) l sto {'40 AIB (DIOXANE SEPARATED) sto 1'40 AA (TCA WASH) M20 1% AAITCA WASH.SONICATED) N=7 F40 AA (DIOXANE SEPARATED) N220 l l l l l l l 2 3 4 5 6 HOURS AFTER '4C AMINO ACID INJECTION 46 in the precipitated protein. Thus about 60% of the labeled amino acids, injected three hours prior to sampling, are bound in the membrane homogenate with an energy greater than can be overcome by the alkali and TCA washes. When labeling was done by injecting 14C-AIB and the membranes were treated in the same manner as those described above, a progressively greater recovery of counts was achieved with each partitioning step. As seen in Figure 6, 55% of the l4C-AIB was unbound as determined by the dioxane separation, and 75% was unbound when the intact membranes were washed with the 10% TCA solution. When the membranes were sonically homogenized and then washed in the TCA and NaOH solutions, the quantity of the l4C—AIB removed from the protein fraction was 90%. This value approaches the expected 100% recovery of a nonmetabolizable, but per- meant, amino acid. For a zero time binding determination 0.02 uC of 3 14 HOH and 0.02 uC of the hydrolysate C-amino acids were mixed with sonically homogenized crop tissue. After twenty- four hours of mixing, the protein was precipitated and washed twice with 5% TCA, once with 1N NaOH, and twice with TCA again; the activity in the protein was then determined. Of the 3H label, 5.35 i 1.41% remained with the protein 14 portion, and 0.082 t 0.014% of the C label was found in the precipitate. It is therefore evident that the wash 47 procedure removes essentially all nonpeptide-linked amino acids, but some 3HOH is retained in the protein fraction. Using membranes which were excised after a three- hour label time (3HOH and hydrOlysate l4 C-amino acids) and a twenty-four hour prolactin treatment, the enhancement of protein synthesis by prolactin was examined. The control and prolactin-treated tissues were separated into two por- tions and sonically homogenized. One portion was used for a total activity determination. The other portion was treated with TCA and NaOH as described in the preceding paragraph; these tissues were then examined for radio- activity. The results (Table 4) indicate an enhanced up- take of label totally, and also the precipitated protein had a significantly higher l4C-label content. The Method gt Increased Protein Synthesis The antibiotics actinomycin D and puromycin were employed to ascertain how prolactin effects an increased protein synthesis. As can be seen in Table 8, actinomycin D counteracted the prolactin—stimulated accumulation of 3HOH and hydrolysate l4 C-amino acids. This inhibition occurred both with the total counts and the washed portion, i.e. the precipitated protein. The puromycin data (Table 9) indicates that this antibiotic is inhibitory only on the 3HOH uptake. No 1 effect on the total 4C-leucine or protein-bound 14C-leucine muouum .pum H mammz h n z mo.vmt ms\zma I O H.mhm.mm n O e.mahe.oe n O Naheea n O we.hmm.e n O\s Ha.hmo.a u Oxa Aho.He~.H n Oxe momm whoa w ea.hhm.m n O mw.Hem.m n O em.hae.m n O hH.Hme.o u O\e mH.Hmm.o u O\a «o~.AsO.H u O\e OCHOOOHIOeH «OB H.Hhe.e~ n O O.Hhm.H~ n O m.HhH.o~ I O no.A~m.o n O\e so.hsm.o n O\a «mo.AeH.H u O\a OCHOOOHIOeH who: zHeueqommenmm+ O zHOmmczHeOa mam.o szuaqomm mama Osman awe: O zHowsozHeum mam.o BZWZBflMMB .momm Och OOAOOOHIOeH :fluomHoum nuflz Q aflohfiocfiuom cam .sfluomaoum .Q Gwomaocwuom Mo uommmw mnall.m OHQMB mo sofiumuomuoocw can mxmumn mg» no muouum .Upm H mcmwz h u z mo.vm« msxsmn I O maheoa n O m.mho.mm n O Naheoa n O SH.AO~.H u O\s mo.aom.o u O\e eeo.umm.a u O\s momm OCOC em.aem.m n O Hm.hoe.~ n O em.ham.m n O m” «mH.hee.H u O\a Hm.Ame.H u O\e «om.het.a u O\a mahoemauuea «Os e.ahm.ea n O o.HAe.HH n O m.HAH.o~ n O «~H.Am~.a u O\e no.hmo.a n O\e «mo.aea.a u O\e OCHODOHIOOH COCO zHeoaqommxmzm~+ zHszomDm meoom zHeoeqomm mnmw qmmgq mmtz zHoszomDm mnoom ezmzeemme .momm cam mawosmalva mo coflumnomuoocfl can mxmums msu co sfluowaonm nuw3 aflomaousm can .sfluomaonm .swomsousm mo pommmw OSBII.m magma _._. ‘wl_ ._ n m“:- 50 as effected by prolactin was observed. Recirculation of the puromycin and its resultant equal inhibition of both lateral portions of the crop are evident by the equal de- crease in accumulation of l4C-leucine in the control counts (puromycin only vs. prolactin only). Prolactin appears to have, at least partially, exerted its effect when admin- istered subsequent to puromycin. Electron Microscopy The initial changes in the pigeon crop epithelium as effected by prolactin were examined by viewing eighteen- hour treated tissues with an electron miscroscope. The prolactin influence can be observed by comparing the photo- micrograph of Figure 7 to that of Figure 8. The thickening of the epithelial layers, i.e. the stratum basalae and the stratum spinosum, is clearly shown. Evident also in the spinosum layer and especially the basalae layer is the treatment effect on cellular size; i.e. a marked increase in response to prolactin is shown. Nuclear material is also accumulated into a denser mass, perhaps indicating the preparation for cell division; this is especially evident in the stratum basalae. . 2‘ n2» Ira-'3' . 26‘-.- was... 157.: cm 1;. M 1.1.21.1 1;. Jere, _ 51 FIGURE 7 Photomicrograph of untreated crOp mucosal epitheliwm, x4400. SD, stratum disjunctum; SS, stratum Spinosum; SB stratum basale; LP, lamina propria. FIGURE 8 Photomicrograph of eighteen hour, 25 ug prolactin-treated pigeon crOp mucosal epithelium, x4400. CHAPTER V DISCUSSION The enhanced uptake of amino acids into the pigeon crop in response to exogenous prolactin is dose-related. Also, there is a nineteen-hour period before more radio- activity of the 14 C-amino acids was found in the prolactin- stimulated vs. the control tissues, and this suggests that other events are occurring prior to the increased amino acid uptake. It is reasonable to assume that certain of these events are prerequisites for the enhanced protein synthesis leading to the crop-sac proliferation. Tata (1963) found an enhanced amino acid uptake following thyroxine administration to the rat liver, and this in- crease was found only after a twenty—six-hour treatment. Increased oxygen consumption (Tata 1963), decreased con— tent in the liver of glycogen (Tata 1963), increased phosphblipid synthesis (Tata 1966), and increased ribo- nucleic acid synthesis (Tata and Windell 1966) all occurred prior to the enhanced amino acid accumulation in response to thyroxine. Further substantiating the view that similar events would occur prior to the increased amino acid uptake in the prolactin-stimulated crop is the 53 54 observation that actinomycin D inhibits amino acid uptake in the crop. This indicates there is an enhanced ribo- nucleic acid synthesis in response to prolactin, and this probably occurs prior to the increased protein synthesis. The AIB T/C ratios greater than 1.0 (Table 4) show that prolactin also stimulates the uptake of this non— metabolizable amino acid, but it is obvious that its en- hanced accumulation cannot depend upon its utilization for incorporation into protein. It is assumed (Noall gt gt. 1957) that the AIB enters the cells under the same influ- ences as some of the metabolizable amino acids. But if the effect of prolactin on the mucosal epithelium is the greater utilization of metabolizable amino acids into pro- tein, then this may account for the enhanced uptakes of both AIB and hydrolysate amino acids. The possibility also exists that prolactin causes a greater rate of amino acid transport apart from a greater rate of protein synthesis. The case for the enhancement of protein synthesis in the pigeon-crop in response to exogenous prolactin is as follows. The maximum recovery of unbound hydrolysate l4C—amino acids from TCA-washed membranes, whether soni- cated or intact, was about 40% (filled square and open circle of Figure 6). This compares well with the 60% retention of the initial counts of hydrolysate l4C-amino acids in the TCA-washed precipitates of sonicated, treated and untreated crop epithelia taken three hours after the 55 labeling (Table 4). A wash with lN NaOH, which should have removed all the amino-acyl-t-RNA-linked amino acids, did not alter the 60% of labeled amino acids retained in the membrane precipitates. It is inferred that this 60% is peptide-linked.. This is a reasonable inference since it was shown that l4C-AIB, which had accumulated in a sig- nificant amount, is almost entirely eluted by the TCA and l4C-AIB and 14C- NaOH washes. Likewise, when both hydrolysate amino acids were added to pigeon blood tg ytttg, a condition under which no protein synthesis would be expected, over 90% of both kinds of labeled amino acids was removed from the precipitated plasma proteins. Finally, the protein precipitates from the prolactin- treated membranes contained 43% more TCA-resistant l4C activity than the control membranes (Table 4). Therefore, it seems evident that prolactin stimulates protein syn- thesis within the pigeon-crop mucosal epithelium. The prolactin-stimulated uptake of.l4C-leucine (a 13% increase) tg ytzg was considerably less than the enhanced uptake of the labeled hydrolysate amino acids (a 41% increase). Since this 41% increase is an average of the enhanced uptakes of the thirteen labeled amino acids, the uptakes of other of the hydrolysate amino acids appear to be more affected by prolactin than leucine. In the tg vivo experiments, the amount of injected labeled amino acids was constant, and the control counts for the 56 leucine and hydrolysate mixtures were 2.77 DPM/mg and 43.3 DPM/mg respectively. The uptake of leucine into the pigeon crop therefore appears to be much less than other amino acids in the hydrolysate mixture. The 3HOH uptake ratios were no different whether determined with the leucine or hydrolysate amino acids; a uniform response to prolactin therefore appears evident. l4C—leucine Since a prolactineenhanced uptake of occurred in the t2 ytttg preparation, it appears that the epithelium alone increases its amino acid accumulation when the crop-sac is exposed to prolactin tg gtyg. Whether an exposure of the epithelium to prolactin entirely tg ytttg will effect an increased amino acid accumulation has not yet been shown since the crop epithelium does not usually remain viable for more than ten hours using this particular tg ytttg technique. And from the tg gtzg studies it appears that nineteen hours is the minimal time necessary for prolactin to effect an increased amino acid accumulation. It is therefore not clear whether prolactin affects the epithelium directly or through some intermediate process, presumably through the more superficial areas of the crop- sac. But the epithelium seems the most likely target tissue since changes were noted earliest in this layer. Actinomycin D apparently inhibits m-RNA synthesis in the nucleus (Reich 1963), and m—RNA is an integral part of the protein synthetic process. Since the 57 prolactin-stimulated protein synthesis is inhibited by actinomycin D, it is apparent that one of the effects of prolactin is on DNA-RNA dependent protein synthesis. Both the accumulation and incorporation of amino acids into protein were inhibited by this antibiotic, and, therefore, it seems that prolactin-stimulated RNA production is essential for both these events. Yet the cause and effect, —‘.l! a t if any exists, of the increased protein synthesis and free amino acid accumulation in the prolactin-stimulated cells remains to be delineated. Also unclear is the mechanism 3 by which the HOH accumulation is inhibited by actinomycin D, although this could be explained partially by the in- hibition of amino acid accumulation, assuming water move- ment is coupled to metabolite fluxes. Puromycin, an antibiotic which inhibits the amino acid transfer from t-RNA to protein, did not inhibit the prolactin-stimulated uptake and incorporation of 14C- leucine. The fact that gross counts in the hormone and calf-serum-treated tissues were fewer indicates that pur- omycin reaches the non-treated crop surface, and label uptake is reduced equally for both crop-membrane surfaces. The prolactin-stimulated uptake in the puromycin-treated tissue means that either the prolactin effect on the crOp is puromycin-insensitive, or puromycin was present in an insufficient quantity to inhibit the effect of prolactin. Sherry and Nicoll (1967) also found only a partial 58 inhibition of the crop-sac response to prolactin when they used either actinomycin D or puromycin. Therefore, it seems possible that the crop-sac epithelium has two types of protein synthesis: one, puromycin—sensitive, and the other puromycin—insensitive, but prolactin-sensitive. It is also possible that prolactin counteracts the effect of puromycin. The mobilization of metabolites preceding the en- hanced protein synthesis may explain the dose-related, time-dependent hydration of the prolactin—stimulated epithelium. Hydration responses are a well—established phenomenon in the proliferative organs of some other spe- cies. Rudolphand and Samuels (1949) demonstrated that ten hours was required before testosterone could effect a water imbibition in the seminal vesicles of castrate rats. Peak hydration levels of the estradiol-stimulated rat uterus were obtained at six hours and after twenty hours of exposure to the hormone (Astwood 1938, Szego and Roberts 1953). This compares well to the brief peak at four hours and the persistent hydration beginning at fifteen hours for the prolactin-stimulated crop mucosa. The four-hour peak was attributed by Szego to an increased capillary permeability and the twenty-hour response to proliferative activities. Within twenty-four hours of prolactin treat— ment, vascularization of the crop-sac wall increases (Dumont 1965). This also was observed in the present study 59 by gross inspection, incidental to tissue preparation.- It should be emphasized that in the repeated experiments when 3HOH and 14 C-amino acids were simultaneously injected, the T/C ratios for 3HOH were greater than 1.0 at four hours, but the T/C ratios for the hydrolysate 14C-amino acids were not. The uptakes of two solutes, 3H-uridine and 22Na+, are probably related to the four-hour hydration, but it is obvious that the amino acids are not. Recently Farese and Schnure (1967) studied the effect of ACTH on 3H-uridine triphosphate uptake in the rat adrenal gland. They found a prolonged uptake beginning at about fifteen hours, but there was a brief peak uptake at four hours following the hormone administration. This, therefore, almost parallels the 3 HOH peak uptake in the prolactin- stimulated pigeon crop in which the four-hour 3H-uridine uptake was also noted. But unlike prolactin, the ACTH was able to elicit an enhanced 3H-uridine accumulation within one-half hour after the hormone treatment. With the prolactin—stimulated crop, the persistent hydration beginning at fifteen hours precedes the enhanced accumulation of the hydrolysate amino acids, beginning at nineteen hours, by too large an interval to look for a direct relationship. However, it is an attractive poss- ibility that the accumulation of the precursors associated with protein synthesis, i.e. nucleotide uptake leading to the formation of RNA, could exPlain the accumulation of L xmm .- _; "a i. 60 3HOH beginning at fifteen hours after prolactin administra— tion. For the times subsequent to nineteen hours, the T/C ratios are significantly greater than 1.0 for both the 3 14C-amino acids, and these could be HOH and hydrolysate related in a causal manner. This relationship is seen also in the dose-response T/C curves of Figures 3 and 4 when samples were taken after 25ug, twenty-four—hour prolactin treatments. The simultaneous increased uptakes of 22Na+ and 3 14 H—uridine occurred while no increased C—amino acid up- take was noted. Although this is not proof that the 22Na+ and 3H—uridine uptakes are related to the four-hour 3HOH peak, this explanation is plausible. Sodium and amino acid transport across the rabbit ilium (Curran gt gt. 1967), Ehrlich cells (Christensen gt gt. 1967), and rabbit red blood cells (Wheeler and Christensen 1967) have been shown to be interdependent, and a sodium—nucleic acid interde- pendent transport is also possible. The osmotic imbalance caused by the influx of sodium and nucleic acids could be the cause of the four—hour water imbibition. The increased sucrose space at twenty-seven hours following prolactin administration was 13%. It is there- fore evident that an increased extracellular space is responsible for at least part of the uptakes as reported in this manuscript. But the increased extracellular space does not account for the magnitude of most of the reported 61 uptakes. 3HOH uptake was also significantly larger than the increased sucrose Space, and, therefore, intracellular expansion in response to prolactin is also evident. The electronmicrographs show quite vividly some changes induced by prolactin in the crOp epithelium. As was also reported by Dumont (1965), the thickening of the stratum spinosum is clearly shown. The enlargement of the stratum basalae cells at eighteen hours was not evident in the twelve-hour photomicrographs of Dumont (1965). This could result from the water imbibition shown to be initi- ated fifteen hours following the prolactin treatment. The stratum disjunctum layer appears unchanged, and Dumont showed it unchanged until the fourth day of hormone stimu- lation. 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H is eliminated from the 14C channel by raising the lower win— dow of the 14C peaked channel. The 3H and 14C variably ‘ quenched standards are then counted and the efficiency of counts calculated; after each standard is counted, the external standard is also counted. The external standard 3 133 H with Ba peaked channel ratio of the counts in the to the counts in the 3H peaked channel is plotted against the efficiency of the corresponding standard. This proce— dure provides a linear 3H efficiency vs. external standard ratio plot; curves for 14C efficiency and 14C in the 3H channel are also obtained. The resulting curves and the instrument settings are illustrated in Figure 9. 3 14 After the H and C counts per minute are printed for an unknown sample, the external standard ratio is also printed. The efficiencies of count are then found from the quench correction plot. The total 14C activity in disintegrations per minute (DPM) in the sample is calculated by dividing the 14C counts per minute (CPM) by the 14C We" .2 “M 69 FIGURE 9 Dual label, 3H and 14C, quench correction curves using an 3 external standard. Channel A is peaked for H, channel B for 14C and channel C for 3H and 1333a 70 ~<\ov Quiz o¢mmlhucm3u cmxmu mnm3 mmHmEMm mmHooem amqmmaquoam mm «ace msHmomzoo "HHH NHazmmmfl mm» mm» mm” mm» mm» mo» 02 02 mm» WON OZ 02 omumowcom mnson owns» HH¢ 74 hHHmN mHHov maumm .mfimcfi .mflmcw .mwmcfl .mfimcw .mfimcw .mwmcw .mwmcw .mflmcw .mmeH OWOOHOCH w NHHvoa vm.a¢m.m v.HHm.mH m.mflo.mm Hm.aon.~ o.HHm.HH H.mwm.mm ha.wbm.m H.Hwo.¢m h.NHHm.oh mv.va.m m.HHm.HN wHHHma H.Hfimm.m H.~Hm.ma o.mw~.am vm.w~m.~ m.HHo.ma w.mwv.am H¢.Hmo.m m.NHh.mH momm ocwocmHIU¢H osflosoHIUvH co Acwuomaoum suw3v cwoheonsm momm ocfiosmHIowa mcdosm I . H Ova so Acauomaoum ocv Gaomsouzm momm wcwosmHIova mcaoom I . 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