0O13-7227/78/1035-1752$O2.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 5 Printed in U.S.A.
Stimulation of Ovine Placental Lactogen Secretion by Arginine Infusion* f S. HANDWERGER4 M. C. CRENSHAW, A. LANSING, A. GOLANDER, T. W. HURLEY,§ AND R. E. FELLOWS^ Departments of Pediatrics, Physiology, and Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT. Arginine has been demonstrated to be a potent stimulus to GH and PRL secretion. To determine the effect of arginine on plasma ovine placental lactogen (oPL) concentrations, arginine (50 g in 350 ml distilled water, pH 7.4) or hypertonic saline of identical volume, osmolality, and pH was infused iv over a 30-min period into nine pregnant ewes, and blood samples from chronic indwelling venous catheters were obtained at frequent intervals before and for 8 h after the infusions. After the infusion of hypertonic saline, plasma oPL concentrations (measured by homologous RIA) decreased 20-50% over 1-2 h and then returned to baseline concentrations. After the infusion of arginine, plasma oPL concentrations also decreased by 20-50% for 1-2 h. However, 2-3 h after the infusion, plasma oPL concentrations in-
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VINE placental lactogen (oPL) is a polypeptide hormone with chemical and biological properties similar to ovine GH (oGH) and ovine PRL (oPRL) (1, 2). Like GH, oPL stimulates weight gain (3), epiphyseal growth (4), and somatomedin production (5) in hypophysectomized rats; like PRL, oPL stimulates lactation in vivo (6) and casein synthesis (6) and iV-acetyl lactosamine synthetase activity (7) in vitro. Although the pattern of oPL secretion during pregnancy has been determined (8-10), the factors regulating its secretion are unknown. Because of the similarities Received January 27,1978. Address reprint requests to: Dr. Stuart Handwerger, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710. * This research was supported by grants from the NIH (HD-07447) and the National Foundation-March of Dimes (1-297). f Presented in part at the 59th Annual Meeting of the Endocrine Society, Chicago, Illinois, June 1977 {Endocrinology 100: A116, 1977). $ USPHS Research Career Development Awardee (HD00065). § Recipient of a Special Research Fellowship (HD05508) from the USPHS. H Present address: Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242.
creased 79-115% (A = 204-700 ng/ml) over preinfusion concentrations in seven ewes and 454% (2930 ng/ml) and 1142% (2042 ng/ml) in two ewes and remained elevated for the remainder of the 8-h interval. When the amount of arginine infused was reduced from 50 to 25 g, an increase in plasma oPL concentrations occurred in only one of five ewes. Plasma oPL concentrations increased by 8-58% after infusions of 50 g alanine or glycine but did not increase after 50 g glutamic acid.
The delayed oPL response to arginine suggests that the increase in plasma oPL concentrations is not caused directly by arginine but rather by changes in the synthesis, secretion, and/or degradation of oPL induced indirectly by arginine. (Endocrinology 103: 1752, 1978)
in the chemical and biological properties of oPL, oGH, and oPRL, we have postulated that oPL secretion may be stimulated by one or more factors which stimulate the secretion of oGH or oPRL. To test this hypothesis, we have investigated the effects of arginine—a stimulus for GH (11) and PRL secretion (12)— on plasma oPL concentrations. Materials and Methods Animals Nine Dorset ewes weighing 47-62 kg were studied between 80 and 135 days of pregnancy. The sheep were housed in individual stalls throughout the experimental period and fed a diet of hay and mixed grains with vitamin and mineral supplements. An indwelling polyvinyl catheter for blood sampling was placed into an iliac vein of each animal at least 5 days before study. The hematocrits of each ewe, tested at biweekly intervals throughout the entire experimental period, were within normal limits. Preparation of amino acid solutions Arginine and alanine were obtained from Eastman Kodak Company, Rochester, NY; glutamic
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STIMULATION OF oPL SECRETION acid and glycine were obtained from Sigma Chemical Company, St. Louis, MO. Fifty grams of each amino acid were dissolved in 350 ml distilled water and the pH was adjusted to 7.4 with concentrated hydrochloric acid. Twenty-five grams of arginine were dissolved in 250 ml deionized water and the pH adjusted to 7.4. Amino acid solutions were prepared within 24 h of infusion, sterilized by Millipore filtration, and transferred to a Viaflex sterile plastic container (Travenol Laboratories, Inc., i Deerfield, IL). The solutions were kept at 4 C until 1 h before infusion. The osmolalities of the 50-g solutions of arginine, glycine, glutamic acid, and alanine were 1110,1305,1424, and 1700 mosm/liter, respectively; the molalities were 0.82,1.90,0.97, and 1.60, respectively. The osmolality of the 25 g arginine solution was 640 mosm/liter. In addition, hypertonic saline solutions of 1100 and 650 mosm/ liter were prepared by dissolving 12.2 g sodium chloride in 350 ml deionized water and 4.8 g sodium chloride in 250 ml deionized water, respectively; the pH of each solution was adjusted to 7.4 with concentrated hydrochloric acid, and sterilized as described above. Experimental procedure The ewes were fasted except for water for 18 h before experiments. On the morning of the experiments, the ewes were placed in separate restraining cages, a polyvinyl catheter was inserted into a jugular vein of each animal, and an infusion of normal saline was begun at a rate of 50 ml/h. Thirty and 60 min later, blood samples (3 ml), designated as —30-min and 0-min samples, were obtained from the iliac vein catheters. The saline infusions were then discontinued and an amino acid solution or hypertonic saline was infused over a 30-min period , using the same catheter. The jugular vein catheter was removed at the end of each infusion and blood samples (3 ml) were collected from the iliac vein catheter 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min after the beginning of each infusion. The plasma samples were frozen at —70 C until assayed. • Each amino acid or hypertonic saline solution was infused into the ewes on separate occasions between 85 and 100 days of pregnancy. Fifty grams of arginine were infused into all nine ewes, 25 g arginine were infused into five ewes, 50 g glycine and glutamic acid were infused into two ewes, and 50 g alanine were infused into three ewes. In control experiments, hypertonic saline of 1100 mosm/liter was infused into four ewes and hypertonic saline of 650 mosm/liter was infused into three. In addition, five ewes were infused a second time with 50 g
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arginine at 110-125 days of pregnancy and one ewe was infused a third time with 50 g arginine at 135 days of pregnancy. Plasma oPL concentrations were measured in all experiments. Plasma oGH concentrations were measured in five ewes receiving 50 g arginine, three receiving 25 g arginine, and two receiving each of the hypertonic saline solutions. Plasma oPRL concentrations were measured in seven ewes receiving 50 g arginine, three receiving 25 g arginine, and two receiving each of the hypertonic saline solutions. Plasma glucose concentrations were measured in six ewes receiving 50 g arginine, four receiving 25 g arginine, and three receiving hypertonic saline solutions. The basal plasma oPL concentration changes with the duration of pregnancy and the absolute concentrations of oPL vary considerably among ewes (8-10). Consequently, to facilitate comparison of the oPL responses to the various infusions, the changes in plasma oPL concentrations during each experiment are expressed as a percentage of the baseline (0 time) concentration. Plasma oGH and oPR concentrations are expressed as nanograms per ml. RIA Plasma oPL concentrations were measured by a specific homologous radioimmunoassay (8) in which oPRL or oGH in concentrations as high as 1000 ng/ml did not displace [125I]oPL. Plasma oGH (13) and oPRL concentrations (14) were measured by homologous RIA using the double antibody method. The oGH and oPRL were provided by the Hormone Distribution Office, NIAMDD, and further purified on Sephadex G-150 (15). The oGH and oPR were iodinated with 125I by the chloramine-T method and antisera to oGH and oPRL were produced in rabbits as described previously for the iodination and production of antisera to hPL (16) and oPL (8). In both assays, serial dilutions of pregnant and nonpregnant sera paralleled the standard curves using purified oGH or oPRL. In the oGH RIA, oPL or oPRL in concentrations of 1000 ng/ml did not displace [125I]oGH from the oGH antisera; and, in the oPRL RIA, oPL or oGH in a concentration of 1000 ng/ml did not displace [125I]oPRL. Other measurements Plasma osmolalities were determined by the freezing point method using a precision osmometer; plasma sodium concentrations were determined by flame photometry. Plasma glucose concentrations were determined by the glucose oxidase method using a Beckman glucose analyzer.
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HANDWERGER ET AL.
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Results Arginine and saline infusions After the infusion of 50 g arginine, plasma oPL concentrations decreased 20-50% and remained below the preinfusion concentrations for 1.5-2 h (Fig. 1). Two to 3 h after the start of the infusion, plasma oPL concentrations increased above the baseline concentrations in all experiments and remained elevated for the remainder of the 8-h period. In studies performed at 85-100 days of pregnancy, plasma oPL concentrations increased to a maximum of 79-115% (A = 204-700 ng/ml) above the preinfusion concentrations in seven ewes and to a maximum of 1142% (A = 2042
FIG. 1. The effects of 50 g arginine or hypertonic saline on plasma oPL concentrations. Fifty grams of arginine were infused into ewes at 85-100 days of pregnancy (A A), 110-125 days of pregnancy (O O), and 135 days of pregnancy (D • ) . In control experiments, hypertonic saline (• • ) of identical volume, osmolality, and pH as the 50 g arginine was infused into ewes at 85-100 days of pregnancy. Each infusion occurred between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration. Baseline plasma oPL concentrations before the arginine infusion were: 337 ± 130 ng/ml (mean ± SD), 85-100 days; 877 ± 208 ng/ml, 110-125 days; and 607 ng/ml, 135 days. The plasma oPL concentrations before the saline infusions were 399 ± 108 ng/ml. The identification number of each animal is given in the left upper corner of each graph.
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ng/ml) and 454% (A = 2930 ng/ml) in two other ewes. When five ewes were again infused with 50 g arginine between 100 and 125 days of pregnancy (Fig. 1), the plasma oPL responses in two were nearly identical to those noted at 85-100 days whereas the maximal oPL responses in three were 25-92% less than those noted earlier in pregnancy. Ewe 50, infused with 50 g arginine at three separate times during pregnancy, had maximal increases in plasma oPL concentrations of 1142% at 91 days of pregnancy, 116% at 116 days, and 99% at 135 days. Plasma oGH concentrations increased by 5-12 ng/ml in three ewes and remained unchanged in two (Fig. 2). Plasma oPRL concentrations increased by 150-500 ng/ml in seven ewes (Fig. 3). In contrast to plasma oPL concentrations, the increase in plasma oGH and oPRL concentrations occurred 0.5-1.5 h after the start of the arginine infusions. After the infusion of hypertonic saline of identical volume, pH, and osmolality as the 50 g arginine, plasma oPL concentrations also decreased 20-50% below the preinfusion concentrations during the first 1.5-2 h (Fig. 1). However, during the remainder of the 8-h period, the plasma oPL responses to hypertonic saline were markedly different from the
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FIG. 2. The effects of 50 g arginine or hypertonic saline on plasma oGH concentrations. Fifty grams of arginine (A A) or hypertonic saline ( • • ) of identical volume, pH, and osmolality were infused into pregnant ewes between 0 and 30 min.
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STIMULATION OF oPL SECRETION
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FIG. 3. The effects of 50 g arginine or hypertonic saline on plasma oPRL concentrations. Fifty grams of arginine (A A) or hypertonic saline ( • • ) of identical volume, pH, and osmolality were infused into pregnant ewes between 0 and 30 min.
FIG. 4. The effects of 50 g arginine (left) or hypertonic saline (right) on plasma osmolality and oPL concentrations, and the effects of 50 g arginine (left) on plasma sodium concentrations. Fifty grams of arginine or hypertonic saline were infused into two ewes between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration.
over the 8 h of observation (Figs. 2 and 3). After the infusion of 50 g arginine, plasma glucose concentrations increased from a baseline of 58 ± 8 mg/dl (mean ± SD) to a maximum of 77 ± 18 mg/dl at 90 min and then returned to baseline concentrations by 120-150 min. After the infusion of hypertonic saline, plasma glucose concentrations did not increase significantly above baseline concentrations. The effects of the infusion of 50 grams of arginine on plasma osmolality and sodium concentrations was determined in two of the arginine studies (Fig. 4). Plasma osmolalities increased from baselines of 286 and 294 mosm/liter to maximums of 310 and 320 mosm/liter 30 min after the start of the infusions and then gradually returned towards the preinfusion osmolalities. Plasma sodium concentrations decreased from 137 and 140 meq/ liter to 126 and 124 meq/liter 30 min after the start of the infusions and then gradually returned toward baseline concentrations. The nadir of the plasma oPL concentrations in both experiments occurred at 30 min. The infusion of hypertonic saline of identical osmolality as the 50 g arginine caused a similar change in plasma osmolalities (Fig. 4).
responses to arginine. In two ewes, plasma oPL concentrations did not increase above Arginine (25 g) preinfusion concentrations. In the remaining After the iv infusion of 25 g arginine, plasma two ewes, the plasma oPL concentrations increased by only 7% and 29%. No changes in oPL concentrations decreased 10-30% during plasma oGH or oPRL concentrations occurred the first 2 h after the start of the arginine
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HANDWERGER ET AL.
1756 FIG. 5. The effect of 25 g arginine on plasma oPL (a) and oPR (b) concentrations. Twenty-five grams of arginine were infused into the pregnant ewes between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration. The baseline oPL concentrations for the five ewes was 318 ± 147 ng/ml (mean ± SD). In control experiments, hypertonic saline of identical volume, osmolality, and pH as the 25 g arginine had no effect on plasma oPL or oPR concentrations.
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infusion (Fig. 5a). Thereafter, plasma oPL concentrations in one animal (ewe 63) increased sharply, reaching a maximum of 165% above the preinfusion concentration at 4 h. In three other ewes, the plasma oPL concentrations increased by 8%, 28%, and 34%. Plasma oGH concentrations increased by only 1-2 ng/ ml whereas plasma oPR concentrations increased by 14, 49, and 195 ng/ml 1-2 h after the initiation of the infusions (Fig. 5b). Plasma glucose concentrations increased from a baseline of 53 ± 14 mg/dl (mean ± SD) to a maximum of 74 ± 16 mg/dl at 90 min and then returned to baseline concentrations by 120-150 min. Glutamic acid, glycine, and alanine The plasma oPL responses to the iv infusions of 50 g glutamic acid, glycine, or alanine are shown in Fig. 6. Glutamic acid caused no increase in plasma oPL concentrations in the two ewes tested. Glycine caused maximal increases in plasma oPL concentrations of 23 and 58%, alanine caused maximal increases of 8%, 43%, and 49%.
Discussion The results of this study demonstrate that the infusion of 50 g arginine is an evocative stimulus to the secretion oPL as well as to the secretion of oPRL and oGH. oPL secretion was stimulated in all 15 experiments in which 50 g arginine were infused. oPRL secretion was stimulated in all seven experiments in
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HOURS FIG. 6. The effects of 50 g glutamic acid, glycine, or alanine on plasma oPL concentrations. Fifty grams of each amino acid were infused into pregnant ewes between 0 and 30 min. The changes in plasma oPL concentrations in each ewe are expressed as a percentage of the baseline (0 time) concentration.
which PRL concentrations were measured and GH secretion was stimulated in three of five ewes. The infusion of 25 g arginine, on the other hand, was not as effective, inasmuch as increases in oPL and oPRL secretion compa-
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STIMULATION OF oPL SECRETION rable to those after the infusion of 50 g arginine were noted in only one ewe and plasma oGH concentrations did not increase above baseline concentrations in any of the ewes. In a small number of ewes, infusions of 50 g alanine or glycine, but not glutamic acid, also stimulated oPL secretion. However, the magnitude of the increases in plasma oPL concentrations in response to alanine and glutamic acid infusions in four of five ewes was less than the magnitude of the increases in response to 50 g arginine, even though approximately twice as many moles of alanine or glycine were infused. Although the infusion of 50 g arginine stimulated oPL, oPRL, and oGH secretion, the pattern of oPL secretion was markedly different than the pattern of either oPRL or oGH secretion. In all experiments, plasma oPL concentrations decreased by 20-50% during the first 1-2 h after the infusion and then increased above preinfusion concentrations by 2-4 h. Increases in plasma oPRL and oGH concentrations occurred 0.5-1 h after the start of the arginine infusions and were not preceded by a decline in plasma concentrations. The magnitude of the increases in plasma oPL and oPRL concentrations varied considerably among the different ewes and there was no correlation between the magnitude of the oPL and oPRL responses. The initial 20-50% decrease in plasma oPL concentrations after the infusions of 50 g arginine, alanine, glutamic acid, and hypertonic saline (1100 mosm/liter) may be due in part to hemodilution resulting from fluid shifts into the vascular space inasmuch as the nadir of the plasma oPL concentration after the infusion of 50 g arginine or hypertonic saline coincided with the peak in plasma osmolality and the nadir of the plasma sodium concentration. The possibility that the infusion of hypertonic solutions of 1100 mosm/liter or greater also inhibit the secretion of oPL cannot be excluded. After the infusion of hypertonic solutions of approximately 650 mosm/ liter (25 g arginine or hypertonic saline), plasma oPL concentrations decreased by greater than 20% in only one of eight experiments.
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The mechanism by which the infusion of 50 g arginine stimulates oPL secretion is unknown. The delay preceding the increase in plasma oPL concentrations suggests that the rise may not be a direct effect of arginine per se but may be secondary to one or more of the other factors modulated by arginine. Inasmuch as arginine has been shown to increase plasma concentrations of GH (11), PRL (12), glucose (17), insulin (17), and glucagon (18), and to decrease plasma concentrations of free fatty acids (19), it is possible that the oPL response to the infusion of arginine may be secondary to changes in one or more of those factors. Although the increase in plasma oGH concentrations preceded the increase in oPL secretion, it is unlikely that GH stimulated the secretion of oPL because an increase in plasma oPL concentrations was observed in two ewes in which arginine failed to stimulate an increase in plasma GH concentrations. Our data, however, do not exclude the possibility that the acute increases in plasma oPL concentrations are due to the increase in plasma PRL concentrations. The increase in basal concentrations of oPL during pregnancy, however, cannot be attributed to PRL since basal plasma oPL concentrations increase markedly during the second half of pregnancy (8-10) whereas plasma PRL concentrations do not increase until just before term (14). Since the increases in plasma glucose concentrations after the infusion of 50 g arginine and of 25 g arginine were similar in magnitude, it is unlikely that the increase in plasma oPL concentrations after the infusion of 50 g arginine is secondary to the modest hyperglycemic response. In previous studies (20, 21), the infusion of arginine into pregnant humans has not been shown to have a consistent effect on plasma hPL concentrations; however, in those studies, blood was sampled over a 90-min period and the dose of arginine used was 25 g. Inasmuch as plasma oPL concentrations are frequently not increased until 2.5-4 h after the start of the infusion of arginine, it is possible that the increase in plasma hPL concentrations may have been missed. Furthermore, since consistent stimulation of oPL secretion occurs only
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HANDWERGER ET AL.
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with the infusion of 50 g arginine, it is possible that the amount of arginine used in pregnant humans was insufficient to elicit a consistent response. Therefore, additional studies are necessary to clarify the effect of arginine on hPL secretion. In summary, the results of this investigation demonstrate that the infusion of 50 g arginine is an evocative stimulus to the secretion of oPL, oPRL, and oGH. Since secretion of all three hormones is stimulated by the infusion of arginine, the results suggest that the secretion of oPL, oPRL, and oGH may be regulated in part by similar mechanisms. Acknowledgments We thank J. Barrett and L. Kodack for their technical assistance.
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References 1. Hurley, T. W., S. Handwerger, and R. E. Fellows, Isolation and structural characterization of ovine placental lactogen, Biochemistry 16: 5598, 1977. 2. Hurley, T. W., F. E. Grissom, S. Handwerger, and R. E. Fellows, Purification and partial characterization of the cyanogen bromide fragments of ovine placental lactogen, Biochemistry 16: 5605, 1977. 3. Hurley, T. W., Doctoral dissertation, Duke University, 1976. 4. Chan, J. S. D., H. A. Robertson, and H. G. Friesen, The purification and characterization of ovine placental lactogen, Endocrinology 98: 65, 1976. 5. Hurley, T. W., L. E. Underwood, S. Handwerger, A. J. D'Ercole, R. Furlanetto, and R. E. Fellows, Ovine placental lactogen induces somatomedin: a possible role in fetal growth, Endocrinology 101: 1635, 1977. 6. Handwerger, S., W. Maurer, J. Barrett, T. W. Hurley, and R. E. Fellows, Evidence for homology between ovine and human placental lactogens, Endocrinol Res Commun 1: 403, 1974. 7. Fellows, R. E., F. F. Bolander, T. W. Hurley, and S. Handwerger, Isolation and characterization of bovine and ovine placental lactogen, In Pecile, A., and E. E. Muller (eds.), Growth Hormone and Related Peptides, Excerpta Medica, Amsterdam-Oxford, 1976, p. 315. 8. Handwerger, S., C. Crenshaw, W. Maurer, J. Barrett,
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T. W. Hurley, A. Golander, and R. E. Fellows, Studies on ovine placental lactogen secretion by homologous radioimmunoassay, J Endocrinol 72: 27, 1977. Handwerger, S., W. F. Maurer, M. C. Crenshaw, T. W. Hurley, J. Barrett, and R. E. Fellows, Development of the sheep as an animal model to study placental lactogen physiology, J Pediatr 87: 1139, 1975. Kelly, P. A., H. A. Robertson, and H. G. Friesen, Temporal pattern of placental lactogen and progesterone secretion in the sheep, Nature 248: 435, 1974. Knopf, R. F., J. W. Conn, S. S. Fajans, J. C. Floyd, E. M. Guntosche, and J. A. Rull, Plasma growth hormone response to intravenous administration of amino acids, J Clin Endocrinol Metab 25: 1140, 1965. Rakoff, J. S., T. M. Siler, Y. N. Sinha, and S. S. C. Yen, Prolactin and growth hormone release in response to sequential stimulation by arginine and synthetic TRF, J Clin Endocrinol Metab 37: 641, 1973. Wallace, A. L. C, and J. M. Bassett, Plasma growth hormone concentrations in sheep measured by radioimmunoassay, J. Endocrinol 47: 21, 1970. Davis, S., L. Reichert, and G. Nisewender, Serum levels of prolactin in sheep as measured by radioimmunoassay, Biol Reprod 4: 145, 1971. Fellows, R. E., and A. D. Rogol, Structural studies on bovine growth hormone. I. Isolation and characterization of the cyanogen bromide fragments, J Biol Chem 244: 1567, 1969. Handwerger, S., and L. M. Sherwood, Human placental lactogen (hPL), In Jaffe, B. M., and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 417. Floyd, J. D., Jr., S. S. Fajans, J. W. Conn, R. F. Knoph, and J. A. Rull, Stimulation of insulin secretion by amino acids, J Clin Invest 45: 1487, 1966. Aguilar-Parada, E., A. M. Eisentraut, and R. H. Unger, Pancreatic glucagon secretion in normal and diabetic subjects, Am J Med Sci 257: 415, 1969. Hertelendy, F., K. Takahashi, L. J. Machlin, and D. M. Kipnis, Growth hormone and insulin secretory responses to arginine in the sheep, pig, and cow, Gen Comp Endocrinol 14: 72, 1970. Tyson, J. E., D. Rabinowitz, T. J. Merimee, and H. Friesen, Response of plasma insulin and human growth hormone to arginine in pregnant and postpartum females, Am J Obstet Gynecol 103: 313, 1969. Tyson, J. E., A. C. Barnes, T. J. Merimee, and V. A. McKusick, Isolated growth hormone deficiency: studies in pregnancy, J Clin Endocrinol Metab 31: 147, 1970.
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Vol. 103, No. 5 Printed in U.S.A.
Stimulation of Ovine Placental Lactogen Secretion by Arginine Infusion* f S. HANDWERGER4 M. C. CRENSHAW, A. LANSING, A. GOLANDER, T. W. HURLEY,§ AND R. E. FELLOWS^ Departments of Pediatrics, Physiology, and Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT. Arginine has been demonstrated to be a potent stimulus to GH and PRL secretion. To determine the effect of arginine on plasma ovine placental lactogen (oPL) concentrations, arginine (50 g in 350 ml distilled water, pH 7.4) or hypertonic saline of identical volume, osmolality, and pH was infused iv over a 30-min period into nine pregnant ewes, and blood samples from chronic indwelling venous catheters were obtained at frequent intervals before and for 8 h after the infusions. After the infusion of hypertonic saline, plasma oPL concentrations (measured by homologous RIA) decreased 20-50% over 1-2 h and then returned to baseline concentrations. After the infusion of arginine, plasma oPL concentrations also decreased by 20-50% for 1-2 h. However, 2-3 h after the infusion, plasma oPL concentrations in-
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VINE placental lactogen (oPL) is a polypeptide hormone with chemical and biological properties similar to ovine GH (oGH) and ovine PRL (oPRL) (1, 2). Like GH, oPL stimulates weight gain (3), epiphyseal growth (4), and somatomedin production (5) in hypophysectomized rats; like PRL, oPL stimulates lactation in vivo (6) and casein synthesis (6) and iV-acetyl lactosamine synthetase activity (7) in vitro. Although the pattern of oPL secretion during pregnancy has been determined (8-10), the factors regulating its secretion are unknown. Because of the similarities Received January 27,1978. Address reprint requests to: Dr. Stuart Handwerger, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710. * This research was supported by grants from the NIH (HD-07447) and the National Foundation-March of Dimes (1-297). f Presented in part at the 59th Annual Meeting of the Endocrine Society, Chicago, Illinois, June 1977 {Endocrinology 100: A116, 1977). $ USPHS Research Career Development Awardee (HD00065). § Recipient of a Special Research Fellowship (HD05508) from the USPHS. H Present address: Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242.
creased 79-115% (A = 204-700 ng/ml) over preinfusion concentrations in seven ewes and 454% (2930 ng/ml) and 1142% (2042 ng/ml) in two ewes and remained elevated for the remainder of the 8-h interval. When the amount of arginine infused was reduced from 50 to 25 g, an increase in plasma oPL concentrations occurred in only one of five ewes. Plasma oPL concentrations increased by 8-58% after infusions of 50 g alanine or glycine but did not increase after 50 g glutamic acid.
The delayed oPL response to arginine suggests that the increase in plasma oPL concentrations is not caused directly by arginine but rather by changes in the synthesis, secretion, and/or degradation of oPL induced indirectly by arginine. (Endocrinology 103: 1752, 1978)
in the chemical and biological properties of oPL, oGH, and oPRL, we have postulated that oPL secretion may be stimulated by one or more factors which stimulate the secretion of oGH or oPRL. To test this hypothesis, we have investigated the effects of arginine—a stimulus for GH (11) and PRL secretion (12)— on plasma oPL concentrations. Materials and Methods Animals Nine Dorset ewes weighing 47-62 kg were studied between 80 and 135 days of pregnancy. The sheep were housed in individual stalls throughout the experimental period and fed a diet of hay and mixed grains with vitamin and mineral supplements. An indwelling polyvinyl catheter for blood sampling was placed into an iliac vein of each animal at least 5 days before study. The hematocrits of each ewe, tested at biweekly intervals throughout the entire experimental period, were within normal limits. Preparation of amino acid solutions Arginine and alanine were obtained from Eastman Kodak Company, Rochester, NY; glutamic
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STIMULATION OF oPL SECRETION acid and glycine were obtained from Sigma Chemical Company, St. Louis, MO. Fifty grams of each amino acid were dissolved in 350 ml distilled water and the pH was adjusted to 7.4 with concentrated hydrochloric acid. Twenty-five grams of arginine were dissolved in 250 ml deionized water and the pH adjusted to 7.4. Amino acid solutions were prepared within 24 h of infusion, sterilized by Millipore filtration, and transferred to a Viaflex sterile plastic container (Travenol Laboratories, Inc., i Deerfield, IL). The solutions were kept at 4 C until 1 h before infusion. The osmolalities of the 50-g solutions of arginine, glycine, glutamic acid, and alanine were 1110,1305,1424, and 1700 mosm/liter, respectively; the molalities were 0.82,1.90,0.97, and 1.60, respectively. The osmolality of the 25 g arginine solution was 640 mosm/liter. In addition, hypertonic saline solutions of 1100 and 650 mosm/ liter were prepared by dissolving 12.2 g sodium chloride in 350 ml deionized water and 4.8 g sodium chloride in 250 ml deionized water, respectively; the pH of each solution was adjusted to 7.4 with concentrated hydrochloric acid, and sterilized as described above. Experimental procedure The ewes were fasted except for water for 18 h before experiments. On the morning of the experiments, the ewes were placed in separate restraining cages, a polyvinyl catheter was inserted into a jugular vein of each animal, and an infusion of normal saline was begun at a rate of 50 ml/h. Thirty and 60 min later, blood samples (3 ml), designated as —30-min and 0-min samples, were obtained from the iliac vein catheters. The saline infusions were then discontinued and an amino acid solution or hypertonic saline was infused over a 30-min period , using the same catheter. The jugular vein catheter was removed at the end of each infusion and blood samples (3 ml) were collected from the iliac vein catheter 30, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min after the beginning of each infusion. The plasma samples were frozen at —70 C until assayed. • Each amino acid or hypertonic saline solution was infused into the ewes on separate occasions between 85 and 100 days of pregnancy. Fifty grams of arginine were infused into all nine ewes, 25 g arginine were infused into five ewes, 50 g glycine and glutamic acid were infused into two ewes, and 50 g alanine were infused into three ewes. In control experiments, hypertonic saline of 1100 mosm/liter was infused into four ewes and hypertonic saline of 650 mosm/liter was infused into three. In addition, five ewes were infused a second time with 50 g
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arginine at 110-125 days of pregnancy and one ewe was infused a third time with 50 g arginine at 135 days of pregnancy. Plasma oPL concentrations were measured in all experiments. Plasma oGH concentrations were measured in five ewes receiving 50 g arginine, three receiving 25 g arginine, and two receiving each of the hypertonic saline solutions. Plasma oPRL concentrations were measured in seven ewes receiving 50 g arginine, three receiving 25 g arginine, and two receiving each of the hypertonic saline solutions. Plasma glucose concentrations were measured in six ewes receiving 50 g arginine, four receiving 25 g arginine, and three receiving hypertonic saline solutions. The basal plasma oPL concentration changes with the duration of pregnancy and the absolute concentrations of oPL vary considerably among ewes (8-10). Consequently, to facilitate comparison of the oPL responses to the various infusions, the changes in plasma oPL concentrations during each experiment are expressed as a percentage of the baseline (0 time) concentration. Plasma oGH and oPR concentrations are expressed as nanograms per ml. RIA Plasma oPL concentrations were measured by a specific homologous radioimmunoassay (8) in which oPRL or oGH in concentrations as high as 1000 ng/ml did not displace [125I]oPL. Plasma oGH (13) and oPRL concentrations (14) were measured by homologous RIA using the double antibody method. The oGH and oPRL were provided by the Hormone Distribution Office, NIAMDD, and further purified on Sephadex G-150 (15). The oGH and oPR were iodinated with 125I by the chloramine-T method and antisera to oGH and oPRL were produced in rabbits as described previously for the iodination and production of antisera to hPL (16) and oPL (8). In both assays, serial dilutions of pregnant and nonpregnant sera paralleled the standard curves using purified oGH or oPRL. In the oGH RIA, oPL or oPRL in concentrations of 1000 ng/ml did not displace [125I]oGH from the oGH antisera; and, in the oPRL RIA, oPL or oGH in a concentration of 1000 ng/ml did not displace [125I]oPRL. Other measurements Plasma osmolalities were determined by the freezing point method using a precision osmometer; plasma sodium concentrations were determined by flame photometry. Plasma glucose concentrations were determined by the glucose oxidase method using a Beckman glucose analyzer.
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Results Arginine and saline infusions After the infusion of 50 g arginine, plasma oPL concentrations decreased 20-50% and remained below the preinfusion concentrations for 1.5-2 h (Fig. 1). Two to 3 h after the start of the infusion, plasma oPL concentrations increased above the baseline concentrations in all experiments and remained elevated for the remainder of the 8-h period. In studies performed at 85-100 days of pregnancy, plasma oPL concentrations increased to a maximum of 79-115% (A = 204-700 ng/ml) above the preinfusion concentrations in seven ewes and to a maximum of 1142% (A = 2042
FIG. 1. The effects of 50 g arginine or hypertonic saline on plasma oPL concentrations. Fifty grams of arginine were infused into ewes at 85-100 days of pregnancy (A A), 110-125 days of pregnancy (O O), and 135 days of pregnancy (D • ) . In control experiments, hypertonic saline (• • ) of identical volume, osmolality, and pH as the 50 g arginine was infused into ewes at 85-100 days of pregnancy. Each infusion occurred between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration. Baseline plasma oPL concentrations before the arginine infusion were: 337 ± 130 ng/ml (mean ± SD), 85-100 days; 877 ± 208 ng/ml, 110-125 days; and 607 ng/ml, 135 days. The plasma oPL concentrations before the saline infusions were 399 ± 108 ng/ml. The identification number of each animal is given in the left upper corner of each graph.
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ng/ml) and 454% (A = 2930 ng/ml) in two other ewes. When five ewes were again infused with 50 g arginine between 100 and 125 days of pregnancy (Fig. 1), the plasma oPL responses in two were nearly identical to those noted at 85-100 days whereas the maximal oPL responses in three were 25-92% less than those noted earlier in pregnancy. Ewe 50, infused with 50 g arginine at three separate times during pregnancy, had maximal increases in plasma oPL concentrations of 1142% at 91 days of pregnancy, 116% at 116 days, and 99% at 135 days. Plasma oGH concentrations increased by 5-12 ng/ml in three ewes and remained unchanged in two (Fig. 2). Plasma oPRL concentrations increased by 150-500 ng/ml in seven ewes (Fig. 3). In contrast to plasma oPL concentrations, the increase in plasma oGH and oPRL concentrations occurred 0.5-1.5 h after the start of the arginine infusions. After the infusion of hypertonic saline of identical volume, pH, and osmolality as the 50 g arginine, plasma oPL concentrations also decreased 20-50% below the preinfusion concentrations during the first 1.5-2 h (Fig. 1). However, during the remainder of the 8-h period, the plasma oPL responses to hypertonic saline were markedly different from the
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FIG. 2. The effects of 50 g arginine or hypertonic saline on plasma oGH concentrations. Fifty grams of arginine (A A) or hypertonic saline ( • • ) of identical volume, pH, and osmolality were infused into pregnant ewes between 0 and 30 min.
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STIMULATION OF oPL SECRETION
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FIG. 3. The effects of 50 g arginine or hypertonic saline on plasma oPRL concentrations. Fifty grams of arginine (A A) or hypertonic saline ( • • ) of identical volume, pH, and osmolality were infused into pregnant ewes between 0 and 30 min.
FIG. 4. The effects of 50 g arginine (left) or hypertonic saline (right) on plasma osmolality and oPL concentrations, and the effects of 50 g arginine (left) on plasma sodium concentrations. Fifty grams of arginine or hypertonic saline were infused into two ewes between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration.
over the 8 h of observation (Figs. 2 and 3). After the infusion of 50 g arginine, plasma glucose concentrations increased from a baseline of 58 ± 8 mg/dl (mean ± SD) to a maximum of 77 ± 18 mg/dl at 90 min and then returned to baseline concentrations by 120-150 min. After the infusion of hypertonic saline, plasma glucose concentrations did not increase significantly above baseline concentrations. The effects of the infusion of 50 grams of arginine on plasma osmolality and sodium concentrations was determined in two of the arginine studies (Fig. 4). Plasma osmolalities increased from baselines of 286 and 294 mosm/liter to maximums of 310 and 320 mosm/liter 30 min after the start of the infusions and then gradually returned towards the preinfusion osmolalities. Plasma sodium concentrations decreased from 137 and 140 meq/ liter to 126 and 124 meq/liter 30 min after the start of the infusions and then gradually returned toward baseline concentrations. The nadir of the plasma oPL concentrations in both experiments occurred at 30 min. The infusion of hypertonic saline of identical osmolality as the 50 g arginine caused a similar change in plasma osmolalities (Fig. 4).
responses to arginine. In two ewes, plasma oPL concentrations did not increase above Arginine (25 g) preinfusion concentrations. In the remaining After the iv infusion of 25 g arginine, plasma two ewes, the plasma oPL concentrations increased by only 7% and 29%. No changes in oPL concentrations decreased 10-30% during plasma oGH or oPRL concentrations occurred the first 2 h after the start of the arginine
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HANDWERGER ET AL.
1756 FIG. 5. The effect of 25 g arginine on plasma oPL (a) and oPR (b) concentrations. Twenty-five grams of arginine were infused into the pregnant ewes between 0 and 30 min. The changes in plasma oPL concentrations are expressed as a percentage of the baseline (0 time) concentration. The baseline oPL concentrations for the five ewes was 318 ± 147 ng/ml (mean ± SD). In control experiments, hypertonic saline of identical volume, osmolality, and pH as the 25 g arginine had no effect on plasma oPL or oPR concentrations.
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infusion (Fig. 5a). Thereafter, plasma oPL concentrations in one animal (ewe 63) increased sharply, reaching a maximum of 165% above the preinfusion concentration at 4 h. In three other ewes, the plasma oPL concentrations increased by 8%, 28%, and 34%. Plasma oGH concentrations increased by only 1-2 ng/ ml whereas plasma oPR concentrations increased by 14, 49, and 195 ng/ml 1-2 h after the initiation of the infusions (Fig. 5b). Plasma glucose concentrations increased from a baseline of 53 ± 14 mg/dl (mean ± SD) to a maximum of 74 ± 16 mg/dl at 90 min and then returned to baseline concentrations by 120-150 min. Glutamic acid, glycine, and alanine The plasma oPL responses to the iv infusions of 50 g glutamic acid, glycine, or alanine are shown in Fig. 6. Glutamic acid caused no increase in plasma oPL concentrations in the two ewes tested. Glycine caused maximal increases in plasma oPL concentrations of 23 and 58%, alanine caused maximal increases of 8%, 43%, and 49%.
Discussion The results of this study demonstrate that the infusion of 50 g arginine is an evocative stimulus to the secretion oPL as well as to the secretion of oPRL and oGH. oPL secretion was stimulated in all 15 experiments in which 50 g arginine were infused. oPRL secretion was stimulated in all seven experiments in
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which PRL concentrations were measured and GH secretion was stimulated in three of five ewes. The infusion of 25 g arginine, on the other hand, was not as effective, inasmuch as increases in oPL and oPRL secretion compa-
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STIMULATION OF oPL SECRETION rable to those after the infusion of 50 g arginine were noted in only one ewe and plasma oGH concentrations did not increase above baseline concentrations in any of the ewes. In a small number of ewes, infusions of 50 g alanine or glycine, but not glutamic acid, also stimulated oPL secretion. However, the magnitude of the increases in plasma oPL concentrations in response to alanine and glutamic acid infusions in four of five ewes was less than the magnitude of the increases in response to 50 g arginine, even though approximately twice as many moles of alanine or glycine were infused. Although the infusion of 50 g arginine stimulated oPL, oPRL, and oGH secretion, the pattern of oPL secretion was markedly different than the pattern of either oPRL or oGH secretion. In all experiments, plasma oPL concentrations decreased by 20-50% during the first 1-2 h after the infusion and then increased above preinfusion concentrations by 2-4 h. Increases in plasma oPRL and oGH concentrations occurred 0.5-1 h after the start of the arginine infusions and were not preceded by a decline in plasma concentrations. The magnitude of the increases in plasma oPL and oPRL concentrations varied considerably among the different ewes and there was no correlation between the magnitude of the oPL and oPRL responses. The initial 20-50% decrease in plasma oPL concentrations after the infusions of 50 g arginine, alanine, glutamic acid, and hypertonic saline (1100 mosm/liter) may be due in part to hemodilution resulting from fluid shifts into the vascular space inasmuch as the nadir of the plasma oPL concentration after the infusion of 50 g arginine or hypertonic saline coincided with the peak in plasma osmolality and the nadir of the plasma sodium concentration. The possibility that the infusion of hypertonic solutions of 1100 mosm/liter or greater also inhibit the secretion of oPL cannot be excluded. After the infusion of hypertonic solutions of approximately 650 mosm/ liter (25 g arginine or hypertonic saline), plasma oPL concentrations decreased by greater than 20% in only one of eight experiments.
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The mechanism by which the infusion of 50 g arginine stimulates oPL secretion is unknown. The delay preceding the increase in plasma oPL concentrations suggests that the rise may not be a direct effect of arginine per se but may be secondary to one or more of the other factors modulated by arginine. Inasmuch as arginine has been shown to increase plasma concentrations of GH (11), PRL (12), glucose (17), insulin (17), and glucagon (18), and to decrease plasma concentrations of free fatty acids (19), it is possible that the oPL response to the infusion of arginine may be secondary to changes in one or more of those factors. Although the increase in plasma oGH concentrations preceded the increase in oPL secretion, it is unlikely that GH stimulated the secretion of oPL because an increase in plasma oPL concentrations was observed in two ewes in which arginine failed to stimulate an increase in plasma GH concentrations. Our data, however, do not exclude the possibility that the acute increases in plasma oPL concentrations are due to the increase in plasma PRL concentrations. The increase in basal concentrations of oPL during pregnancy, however, cannot be attributed to PRL since basal plasma oPL concentrations increase markedly during the second half of pregnancy (8-10) whereas plasma PRL concentrations do not increase until just before term (14). Since the increases in plasma glucose concentrations after the infusion of 50 g arginine and of 25 g arginine were similar in magnitude, it is unlikely that the increase in plasma oPL concentrations after the infusion of 50 g arginine is secondary to the modest hyperglycemic response. In previous studies (20, 21), the infusion of arginine into pregnant humans has not been shown to have a consistent effect on plasma hPL concentrations; however, in those studies, blood was sampled over a 90-min period and the dose of arginine used was 25 g. Inasmuch as plasma oPL concentrations are frequently not increased until 2.5-4 h after the start of the infusion of arginine, it is possible that the increase in plasma hPL concentrations may have been missed. Furthermore, since consistent stimulation of oPL secretion occurs only
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with the infusion of 50 g arginine, it is possible that the amount of arginine used in pregnant humans was insufficient to elicit a consistent response. Therefore, additional studies are necessary to clarify the effect of arginine on hPL secretion. In summary, the results of this investigation demonstrate that the infusion of 50 g arginine is an evocative stimulus to the secretion of oPL, oPRL, and oGH. Since secretion of all three hormones is stimulated by the infusion of arginine, the results suggest that the secretion of oPL, oPRL, and oGH may be regulated in part by similar mechanisms. Acknowledgments We thank J. Barrett and L. Kodack for their technical assistance.
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References 1. Hurley, T. W., S. Handwerger, and R. E. Fellows, Isolation and structural characterization of ovine placental lactogen, Biochemistry 16: 5598, 1977. 2. Hurley, T. W., F. E. Grissom, S. Handwerger, and R. E. Fellows, Purification and partial characterization of the cyanogen bromide fragments of ovine placental lactogen, Biochemistry 16: 5605, 1977. 3. Hurley, T. W., Doctoral dissertation, Duke University, 1976. 4. Chan, J. S. D., H. A. Robertson, and H. G. Friesen, The purification and characterization of ovine placental lactogen, Endocrinology 98: 65, 1976. 5. Hurley, T. W., L. E. Underwood, S. Handwerger, A. J. D'Ercole, R. Furlanetto, and R. E. Fellows, Ovine placental lactogen induces somatomedin: a possible role in fetal growth, Endocrinology 101: 1635, 1977. 6. Handwerger, S., W. Maurer, J. Barrett, T. W. Hurley, and R. E. Fellows, Evidence for homology between ovine and human placental lactogens, Endocrinol Res Commun 1: 403, 1974. 7. Fellows, R. E., F. F. Bolander, T. W. Hurley, and S. Handwerger, Isolation and characterization of bovine and ovine placental lactogen, In Pecile, A., and E. E. Muller (eds.), Growth Hormone and Related Peptides, Excerpta Medica, Amsterdam-Oxford, 1976, p. 315. 8. Handwerger, S., C. Crenshaw, W. Maurer, J. Barrett,
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T. W. Hurley, A. Golander, and R. E. Fellows, Studies on ovine placental lactogen secretion by homologous radioimmunoassay, J Endocrinol 72: 27, 1977. Handwerger, S., W. F. Maurer, M. C. Crenshaw, T. W. Hurley, J. Barrett, and R. E. Fellows, Development of the sheep as an animal model to study placental lactogen physiology, J Pediatr 87: 1139, 1975. Kelly, P. A., H. A. Robertson, and H. G. Friesen, Temporal pattern of placental lactogen and progesterone secretion in the sheep, Nature 248: 435, 1974. Knopf, R. F., J. W. Conn, S. S. Fajans, J. C. Floyd, E. M. Guntosche, and J. A. Rull, Plasma growth hormone response to intravenous administration of amino acids, J Clin Endocrinol Metab 25: 1140, 1965. Rakoff, J. S., T. M. Siler, Y. N. Sinha, and S. S. C. Yen, Prolactin and growth hormone release in response to sequential stimulation by arginine and synthetic TRF, J Clin Endocrinol Metab 37: 641, 1973. Wallace, A. L. C, and J. M. Bassett, Plasma growth hormone concentrations in sheep measured by radioimmunoassay, J. Endocrinol 47: 21, 1970. Davis, S., L. Reichert, and G. Nisewender, Serum levels of prolactin in sheep as measured by radioimmunoassay, Biol Reprod 4: 145, 1971. Fellows, R. E., and A. D. Rogol, Structural studies on bovine growth hormone. I. Isolation and characterization of the cyanogen bromide fragments, J Biol Chem 244: 1567, 1969. Handwerger, S., and L. M. Sherwood, Human placental lactogen (hPL), In Jaffe, B. M., and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 417. Floyd, J. D., Jr., S. S. Fajans, J. W. Conn, R. F. Knoph, and J. A. Rull, Stimulation of insulin secretion by amino acids, J Clin Invest 45: 1487, 1966. Aguilar-Parada, E., A. M. Eisentraut, and R. H. Unger, Pancreatic glucagon secretion in normal and diabetic subjects, Am J Med Sci 257: 415, 1969. Hertelendy, F., K. Takahashi, L. J. Machlin, and D. M. Kipnis, Growth hormone and insulin secretory responses to arginine in the sheep, pig, and cow, Gen Comp Endocrinol 14: 72, 1970. Tyson, J. E., D. Rabinowitz, T. J. Merimee, and H. Friesen, Response of plasma insulin and human growth hormone to arginine in pregnant and postpartum females, Am J Obstet Gynecol 103: 313, 1969. Tyson, J. E., A. C. Barnes, T. J. Merimee, and V. A. McKusick, Isolated growth hormone deficiency: studies in pregnancy, J Clin Endocrinol Metab 31: 147, 1970.
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