Release of Pituitary Growth Hormone by Prostaglandins and Dibutyryl Adenosine Cyclic 3':5'-Monophosphate in the Absence of Protein Synthesis

Proceedings of the National Academy of Sciences Vol. 67, No. 3, pp. 11t2-1179, November 1970 Release of Pituitary Growth Hormone by Prostaglandins and Dibutyryl Adenosine Cyclic 3':5'-Monophosphate in the Absence of Protein Synthesis Robert M. MacLeodt and Joyce E. Lehmeyer DEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY OF VIRGINIA SCHOOL OF MEDICINE, CHARLOTTESVILLE, VA. 22901 Communicated by Dietrich Bodenstein, July 24, 1970 Abstract. Effects of prostaglandins on the incorporation of [4,5-3Hlleucine into growth hormone and its subsequent release into the incubation medium were studied. Incubation of rat anterior pituitary glands with 10- M prostaglandin PGE1 in tissue culture medium 199 for 7 hr caused a 40-300% increase in the release of labeled growth hormone into the incubation medium. PGE1 at 10-8 M increased growth hormone synthesis but not release. At 10-6 M, PGE2 had effects similar to PGE1; PGA1 increased growth hormone synthesis- but not release. PGF2a was without effect on either synthesis or release of growth hormone. Prolactin synthesis and release were not affected by prostaglandins. All of the prostaglandins, at 10-4 M, increased adenyl cyclase activity in the pituitary gland but phosphodiesterase activity was unaltered. Dibutyryl cyclic AMP, with or without caffeine, caused an up to 300% increase in labeled growth hormone release. No consistent effect of prolactin was observed. If potassium concentration was increased 10-fold, a 215% increase in growth hormone release was observed. A combination of hypertonic potassium and 10-6 M PGE1 increased growth hormone release 325%, suggesting that potassium and prostaglandins act by independent mechanisms. Addition of theophylline to pituitary gland, incubated in vitro, increased both the synthesis and release of growth hormone. Although fluoride greatly stimulated growth hormone release, it completely inhibited the incorporation of leucine into the hormone. Similarly, puromycin inhibited synthesis of growth hormone but did not block release induced by prostaglandin, dibutyryl cyclic AMP, theophylline, or fluoride. Prostaglandins increase pituitary adenyl cyclase activity and, presumably via cyclic AMP, increase growth hormone release, independently of protein synthesis. Hormonal secretions of the anterior pituitary gland are thought to be regulated by agents released by the hypothalamus into the hypophyseal portal blood vessels leading to the gland.1 The agents or neurohormones that control growth hormone synthesis are not well characterized2 and very little is known about the mechanism by which they affect release of the hormone. The rapidity with which neurohormones cause growth hormone release suggests that stored hormone is discharged from the cell and may directly or indirectly stimulate the synthesis of new hormone. Neurohormones have been shown to affect the 1172

VOL. 67, 1970 PROSTAGLANDINS AND HORMONE RELEASE 1173 pituitary gland, steroidogenesis in the adrenal gland35, lipolysis, and adipose tissue.6 7 These processes are influenced by cyclic AMP generated in the tissues by specific hormones. Prostaglandins, among other substances present in the hypothalamus, alter the intracellular level of cyclic AMP in other tissues.8'9 Materials and Methods. Mature female (180-200 g) and male (200-230 g) Sprague- Dawley rats were obtained from Flow Research Animals, Dublin, Va. Prostaglandins El, E2, F2,, and Al were gifts of Dr. John E. Pike of the Upjohn Co. Incorporation of [4,5-8H]leucine into prolactin and growth hormone was studied by incubating bisected pituitary glands in 0.5 or 1.4 ml of tissue culture Medium 199 containing 5-10,Ci of the isotope. Flasks were incubated for 7 hr at 370C in a Dubnoff shaker under 95% 02-5% CO2. Pituitary glands were homogenized in 0.5 ml of 50 mm phosphate (ph 7.2) with a glass homogenizer fitted with a Teflon pestle. Homogenates were frozen and thawed three times to lyse all granules. Duplicate aliquots (50,ul) were then electrophoresed on polyacrylamide gell 0" with the sample, stacking, and separating gel system of Jones et al.12 The incubation medium was separated on gels but the 50- ;41 aliquots were "top-loaded" using the system described by Reisfeld et al."3 The proteins on the gels were stained with amido black, and quantitated by means of a Canalco microdensitometer with an integrator. The patterns produced by the rat pituitary hormones were identified by comparing their mobility with purified preparations of prolactin and growth hormone. Our results are in excellent agreement with Jones et al.2, who eluted these proteins from the polyacrylamide gels and established their identity by bioassay. Adenyl cyclase activity was determined by the method of Zor et al.,'4 protein by the method of Lowry et al.15 Cyclic AMP phosphodiesterase activity was assayed by the method of Butcher and Sutherland.'6 Results. The effect of prostaglandin PGE1 on the incorporation of [4,5-3H]- leucine into growth hormone in vitro is presented in Fig. 1. The addition of 10- M PGE, increased the growth hormone released into the incubation medium 165%; normal amounts of the hormone were maintained within the pituitary gland. At a concentration of 10-7 iv PGE1, labeled growth hormone 2400 Pituitary Gland Incubation Medium Total 240 ax 1400, ~100c. 600 200 Control 10-6 10-7 10-8 Control 106 1 0- i-8 Control 10-6 10-7 1o-8 Molar concentration of prostaglandin PGE1 FIG. 1. Effect of prostaglandin PGE, on the incorporation of [4,5-3H]leucine into growth hormone. Four hemi-anterior pituitary glands were incubated in four flasks containing 1.4 ml of tissue culture medium 199, 10 MCi of [4,5-3H]leucine, and prostaglandin for 7 hr at 370C; 3 flasks/group.

1174 BIOCHEMISTRY: MAcLEOD AND LEHMEYER PROC. N. A. S. in the medium increased 40%, and only slightly within the tissue. Although growth hormone release was inhibited by 10-8 1\1 PGE1, the synthesis of the hormone in the gland increased. The data presented in Table 1 show that PGE1 and PGE2 increased the release TABLE 1. Effect of various prostaglandtns on the incorporation of [4,5-H]leucine into growth hormone. Absorbance Radioactivity units per No. of.- (cpm/mg pituitary)--- mg pituitary deter- Pituitary Incubation Incubation Prostaglandin minations gland medium medium Control 6 1241 i 63 385 + 36 9.9 PGE, 10-' M 4 1103 + 56 556 i 37* 25.3t PGE2 10-6 M 4 1091 + 22t 557 =1 48t 25.6t PGA10O-M 4 1562+48* 370±30 11.2 PGF?, 10-6 M 4 1054 ±4 49 282 ±A 40 11.8 Values are means ±t SE. * P < 0.01; t P < 0.05 with respect to control value. of newly synthesized hormone 44%, while causing a slight decrease in labeled hormone within the gland. Although PGA1 did not cause the release of labeled growth hormone into the incubation medium, it significantly increased the incorporation of radioactive leucine into the pituitary growth hormone. PGF2< did not increase growth hormone synthesis or release. M1icrodensitometric determinations showed that only PGE1 and PGE2 increased hormone release. Since prostaglandins influence the cyclic AMP concentration in certain tissues, we determined whether adenyl cyclase activity in the pituitary gland was affected by prostaglandins. Table 2 shows that the addition of PGE1, PGA1, and PGF2< increased adenyl cyclase activity in the anterior pituitary gland. TABLE 2. Effect of prostaglandins on the formation of cyclic ['2P]AMP from [a-32p]atp in anterior pituitary glands. Picomoles cyclic [32P]AMP per gland per 60 m in Control PGE, PGF2a PGA, Expt. 1 0.66 ± 0.23 2.31 ±t 0.04*...... Expt. 2 3.53 ± 0.91... 9.31 ±t 2.60t 8.03 ± 1.87t The prostaglandin (6 pg) was incubated 60 min at 37 C with a whole anterior pituitary gland in 40 mm Tris HCl (ph 7.8) containing 3.5mM Mg2 +, 10 mm theophylline, 0.15 mg albumin, 20 mm ATP, 1 uci [a82p]atp, 1.3 mm cyclic AMP, 7 mm PEP, and 4,units pyruvate kinase in a volume of 0.15 ml. The cyclic 132P]AMP was isolated by thin-layer chromatography (Expt. 1) or by separation on a Dowex 50 column (Expt. 2). PEP, phosphoenolpyruvate. * P < 0.005; t P < 0.05. Incubation of pituitary glands for 2 hr with 10-5 M PGE1 in tissue culture medium 199 had no effect on phosphodiesterase activity. Many attempts to show that the sodium salt of cyclic AMP, in the absence of caffeine, influenced growth hormone synthesis were unsuccessful. The results presented in Table 3 show that dibutyryl cyclic AMP caused a consistent increase in growth hormone release: 5 mm produced increases of 220 and 250% and 0.1 mm produced a 180% increase in newly synthesized growth hormone. The fact that radioactive growth hormone did not accumulate within the gland in the

VOL. 67, 1970 PROSTAGLANDINS AND HORMONE RELEASE 1175 TABLE 3. Effect of dibutyryl cyclic AMP on the incorporation of [4,5-3H]leucine into prolactin and growth hormone. Absorbance Radioactivity units GH (cpm/mg pituitary) per mg Prolactin I - Growth hormone--_ pituitary Pituitary Incubation Pituitary Incubation Incubation gland medium gland medium medium Control 154 i 42 348 :1: 74 2589 i 127 677 i 105 9.6 Dibutyryl cyclic AMP 0.1 mm 157 i 18 473 4± 15 2397 i 124 1822 i: 201* 19.9 Dibutyryl cyclic AMP5mM 96 4± 17 376 ± 16 1291 4± 18* 2221 i 111* 22.1 Each group comprised of four flasks containing four pituitary gland halves from male rats, 1.4 ml of medium 199, and 10 uci [4,53H]leucine; incubated 7 hr. *P <.01. presence of dibutyryl cyclic AMP is taken to indicate that the cyclic nucleotide was acting primarily on growth hormone release and not on its synthesis. No consistent effect of dibutyryl cyclic AMP on prolactin release was observed. The data in Table 4 give additional evidence that cyclic AMP acts primarily TABLE 4. Effect of theophylline and NaF on the synthesis and release of growth hormone and prolactin. Radioactivity Absorbance (cpm/mg pituitary) - units per '_ ~Prolactin _- - Growth hormone - mg pituitary Pituitary Medium Pituitary Medium Medium Control... 701 ± 86 3196 ± 265 1362 ± 132 17.6 ±t 0.9 Theophylline 10 mm... 719 ± 141 2847 ± 239 2109 ±- 102* 38.9 ±4 3.9* NaF 10 mm... 7 ± 2 76 ± 5 65 ±t 6* 37.0 ± 6.1* Single pituitary glands were incubated in 0.5 ml of medium 199 containing 5 ACi of [4,5-3H]leucine. Values are means 4 SE. *P < 0.01. on release of the hormone. Theophylline, an inhibitor of phosphodiesterase, produced a significant increase in growth hormone release measured by microdensitometric and by isotopic methods. NaF greatly increased growth hormone release but completely inhibited the synthesis of growth hormone. The effects of theophylline and fluoride on growth hormone synthesis and release are presented in Fig. 2. NaF at 10-2 M increased release 130% but almost completely inhibited the incorporation of leucine into growth hormone. Lower concentrations of fluoride were less effective in stimulating release of growth hormone and inhibiting hormone synthesis. Theophylline increased growth hormone release 130, 170, and 60% when pituitary glands were incubated in the presence of 10-2, 10-3, or 10-4 M theophylline; synthesis was stimulated by 65, 110, or 15%. Table 5 shows that theophylline, PGE1, and dibutyryl cyclic AMP increased the incorporation of leucine into growth hormone; increased the total amount of growth hormone released into the incubation medium; and decreased the amount remaining in the gland. The addition of pyromycin to flasks containing these substances inhibited the incorporation of leucine into growth hormone but did

1176 BIOCHEMISTRY: MAcLEOD AND LEHMEYER PROC. N. A. S. C 0 2000. X 40 1500- "30 a: 2~~~~~~~~~~~~~~0 0 100 O -20 c 0 T I 50/ -10 Or -2 1 0-3 '-4-2 -3-4 0L cc~~~~~~~~~~~~~~ M SODIUM FLUORIDE M THEOPHYLLINE FIG. 2. Effect of various concentrations of NaF and theophylline on the synthesis and release of growth hormone. Two hemi-anterior pituitary glands were incubated as described in Fig. 1 in six flasks containing 0.5 ml of tissue culture medium 199, 5,uCi of [4,5-3H]leucine, and the indicated amounts of NaF or theophylline. The solid lines refer to the absorbance units of growth hormone in the incubation medium and the dashed lines refer to the radioactive growth hormone in the incubation medium. The shaded bars refer to the mean i SE of control flasks; 3 flasks/group. not inhibit release of the hormone. Additionally, puromycin did not inhibit the fluoride-induced increase in growth hormone release. When potassium concentration was made hypertonic, growth hormone release was significantly increased.'7 Fig. 3 shows that the addition of 10-6 M PGE1 to Medium 199 increased growth hormone in the incubation medium 175%; also that increasing the potassium concentration 10-fold, to 59.4 mm, caused a 215% increase in hormone release. The combination of increased potassium concen- TABLE 5. Effect of puromycin on the synthesis and release of growth hormone. Incorporation of [4,5-3H]leucine Absorbance (cpm/mg) -~ - (units/mg) Pituitary Incubation Pituitary Incubation gland medium gland medium Control 4336 4+ 383 2120 i 180 51.6 i 4.6 30.8 :1 3.3 Puromycin 0.2 mm 21 i 5 27 i 15 57.0 37.6 Theophylline 10 mm 4161 i 774 4610 + 683 43.2 53.1 Theophylline + puromycin 9± 8 21+ 5 23.6 44.1 PGE, 1 IM 5408 + 225 2701 i 395 29.0 33.8 PGE, + puromycin 8 i 3 12 i 3 24.6 43.4 Dibutyryl cyclic AMP 1 mm 2595 i 517 4502 i 373 29.4 41.0 Dibutyryl cyclic AMP + puromycin 10 + 3 21 ± 5 24.8 51.3 NaF 10 mm 61 +t 7 87 + 39 22.0 40.0 NaF + puromycin 12 +- 4 10 4-5 25.6 40.5 Two halves of bisected pituitary glands were incubated in 0.5 ml of Medium 199 containing 5 jsci of 14.5-3H]leucine, the indicated compound, and/or puromycin for 7 hr.

VOL. 67, 1970 PROSTAGLANDINS AND HORMONE RELEASE 1177 2600 FIG. 3. Effect of pros- 2200 taglandins and cations on III the incorporation of [4,5-1800 pituitary 3H]leucine into growth hor- TuitaryP mone. Four hemi-anterior 1400 1 pituitary glands were incubated using conditions described in Fig. 1 except that 1000. some flasks contained 10 times as much K+ or Ca++ as 60* medium in the control medium; 3 flasks/group. Ordinate is cpm/mg pituitary. 200- Control PGE1 K+ PGE1 Ca`+ PGE1 K+ PGE1 K+ Ca++ Ca++ K+ Ca++ tration and prostaglandin increased growth hormone release 325%. Calcium, 5.1 mm, had no effect on growth hormone release in the presence or absence of prostaglandin, although the addition of Ca2+ at elevated K+ and PGE1 concentrations increased radioactive growth hormone released into the medium 395%. PGE1 had no significant affect on total growth hormone synthesis, nor did its addition influence the potentiating affect of K+ on hormone synthesis. The incorporation of [4,5-3H]leucine into growth hormone was reduced in potassium-free incubation medium (Table 6); however, the addition of 10-6 1\1 PGE1 to buffer with or without K+ increased growth hormone release. TABLE 6. Effect of potassium ion on the prostaglandin-induced increase of growth hormone synthesis and release. Radioactivity Absorbance units _ ~~(cpm/mg pituitary) - per mg pituitary Pituitary Incubation Incubation Buffer gland medium medium Complete KRB 1736 4+ 96 325 + 22 10.2 KRB minus K+ 667 + 24 170 + 21 13.5 Complete KRB with PGE1 1737 i 92 696 + 46* 25.9* KRB minus K+ with PGE1 1029 ± 99* 314 ± 13* 27.6* Pituitary glands from female rats were incubated in Krebs-Ringer bicarbotmite buffer containing 1.8 mg/ml glucose, a complete essential and nonessential amino acid mixture (Microbiological Associates), and 10 fci of [4,5-3H]leucine for 7 hr at 370C. Prostaglandin PGE1, 10-6 M. Values are means ± SE. *P <.01 Discussion. The ubiquitous distribution of prostaglandins and their many effects on tissues were discussed by Bergstr6m et al.9, but it has only recently been demonstrated that endocrine function is influenced by these substances. The in vivo injection of PGE1 and PGE2, but not P'GFia nor PGF2a, stimulated ACTH release."8"19 Flack28 showed that corticosteroidogenesis is increased by prostaglandin in vitro. In thyroid slices, PGE increased proteolysis2' and the levels of cyclic A'IiP in thyroid,22 lung, spleen, diaphragm and whole fat pads.7 Zor et al.'4 showed that adenyl cyclase activity was increased in rat pituitary glands after prostaglandin addition, and resulted in increased levels of cyclic AMP.

1178 BIOCHEMISTRY: MAcLEOD AND LEHMEYER PROC. N. A. S. We confirm the findings of Zor et al.14 and show that PGE1, PGE2, and PGA1 increase adenyl cyclase activity in the anterior pituitary gland. The release of growth hormone was significantly increased by prostaglandins PGE1 and PGE2 but not by PGA1 or PGF2a. A possible physiological function for the prostaglandins is suggested by the finding that the dibutyryl ester of cyclic AMP greatly increased growth hormone but not prolactin release. TSH23 and ACTH24 have also been shown to be released by dibutyryl cyclic AMP. Prostaglandins apparently have some degree of specificity with regard to pituitary hormone release because prolactin and luteinizing hormone26 are not affected by these agents. Since prostaglandins increase adenyl cyclase activity in the pituitary gland, the cyclic nucleotide produced may increase the release of ACTH, TSH, and GH. Substances that are capable of affecting adenyl cyclase or phosphodiesterase activity in the pituitary also alter hormone release. The in vitro addition of theophylline increased GH release, a result previously found in Vivo.26 Additionally, NaF, an activator of adenyl cyclase but also an inhibitor of glycolysis, produced a large increase in GH release but completely inhibited the incorporation of leucine into the hormone. This is evidence that fluoride was affecting the release of growth hormone directly and not the synthesis of new molecules of growth hormone, and that, since NaF inhibits energy derivation from the glycolytic metabolism of glucose, the release of growth hormone is a minimal- or nonenergy dependent process. That synthesis of new growth hormone is not requisite for release of the hormone has been conclusively demonstrated by the findings that puromycin completely inhibited the synthesis of growth hormone without interfering with the increase in growth hormone release induced by prostaglandin, cyclic AMP, theophylline, or fluoride. The release of luteinizing hormone was not dependent upon synthesis of the hormone,27 nor was release of TSH.28'29 Subsequent to the drug-induced decrease in pituitary growth hormone concentration, an increase in hormone synthesis occurred. Although the mechanisms governing the synthesis of growth hormone are poorly understood, we would speculate that the pituitary gland is capable of detecting a decrease in growth hormone content in cells in which the hormone is synthesized, resulting in a compensatory increase in the synthesis of hormone. Previously, we reported on the selective effects of various ions on growth hormone and prolactin release. 17 Hypertonic amounts of potassium released growth hormone but did not affect prolactin. Conversely, calcium increased prolactin release but had no affect on growth hormone release. Although prostaglandins are known to increase cation flux in several tissues,-3io, the current data show that the incubation of pituitary glands, in media hypertonic with respect to potassium and/or calcium, with prostaglandin PGE1 increased growth hormone release to a greater extent than did PGE1 or the cations separately. These substances probably act independently to release growth hormone. The addition of epinephrine in vitro to several tissues increased adenyl cyclase activity;32-35 however, it has no affect on this enzyme in pituitary tissue. It is probable that catecholamines inhibit prolactin release and synthesis36 by a

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