0013-7227/91/1294-1791$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 4 Printed in U.S.A.

Effect of Antiserum to Rat Growth Hormone (GH)Releasing Factor on Physiological GH Secretion in the Female Rat* MASAMI ONO, NOBUHIRO MIKIf, AND HIROSHI DEMURA Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan

of 1 Mg/kg BW after the last blood sampling, stimulated GH release to a peak level of 153 ± 37 ng/ml in the control rats. Administration of GRF antiserum caused a profound suppression of both pulse and trough components of GH secretion. This effect occurred rapidly, within 15 min after injection of antiserum, and GH secretion decreased uniformly to very low levels (3.4 ± 0.1 ng/ml), with little or no fluctuation throughout the observation period. GRF antiserum also abolished the synthetic rat GRF-induced GH release, indicating sufficient potency of immunoneutralization. These results demonstrate that both GH pulses and troughs are dependent upon hypothalamic GRF in normal female rats, thereby substantiating earlier observations in male rats which demonstrated the physiological role of GRF in GH secretion. (Endocrinology 129: 1791-1796, 1991)

ABSTRACT. To further study the physiological role of GHreleasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH secretion in the normal female rat. Two groups of six conscious female rats were passively immunized with either nonimmune rabbit serum (NRS) or antirat GRF serum via a chronic indwelling atrial catheter. The secretory profiles of GH were observed by collecting blood samples at 15-min intervals for 1 h before and 4 h after administration. The NRS-treated rats showed a characteristic female pattern of spontaneous GH secretion. GH pulses were of low amplitude (mean ± SEM, 26.8 ± 2.4 ng/ml) and occurred irregularly at a frequency of 4.2 ± 0.2/5 h, while interpeak trough levels of GH were relatively high, with nadir values of 8.6 ±0.7 ng/ml. Synthetic rat GRF, given iv at a dose

S

ECRETION of GH by the anterior pituitary gland is under dual control of the two hypothalamic peptides, GH-releasing factor (GRF) (1) and somatostatin (SRIF) (2). GH is secreted in a pusatile pattern in many species of animals (3). In male rats, spontaneous GH pulses are of high amplitude and occur regularly at 3- to 4-h intervals, and interpulse GH troughs are very low (4). GRF generates spontaneous GH bursts. This finding was first observed in male rats by Wehrenberg et al. (5), who demonstrated blockade of episodic GH surges by a monoclonal antibody against rat GRF (rGRF). We later confirmed their findings using polyclonal antibodies and also demonstrated that GRF antiserum inhibited GH release induced by an a-adrenergic agent (clonidine) or an enkephalin analog (FK33-824) (6, 7). In contrast, SRIF antiserum does not inhibit spontaneous GH surges, but consistently raises interpeak GH troughs (8-14), which suggests that GH troughs are under a tonic inhibition by SRIF secretion. More recent studies in rats Received April 8, 1991. * This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, and Culture. t To whom requests for reprints should be addressed.

have demonstrated an inhibitory influence of SRIF on GRF secretion within the hypothalamus (15-17). Most of these studies were performed in rats (5-13, 15-17), and all studies in rats used male rats. However, the secretory pattern of GH in rats shows striking sexual dimorphism. In female rats, GH surges are of lower amplitude and occur irregularly and more frequently, whereas GH troughs are higher than those in male rats (18-21). This difference in GH secretory pattern may result at least in part from a sexually dimorphic mode of secretion of hypothalamic GRF and/or SRIF. We thought it important to determine first whether GRF plays a physiological role in GH secretion in female rats as it does in male rats. A few studies have demonstrated inhibition by GRF antiserum of pharmacologically induced GH release in female rats (22, 23). However, the effect of GRF antiserum on physiological GH secretion has not yet been reported in female rats. The aim of the present study was to investigate the effect of passive immunization with rabbit antiserum to GRF on GH secretion in conscious cycling female rats. The anti-GRF serum used in this study was previously demonstrated to potently immunoneutralize endogenous and exogenous GRFs in male rats (7, 17).

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GRF: PHYSIOLOGICAL ROLE IN FEMALE RATS

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Materials and Methods Animals Female Sprague-Dawley rats were obtained at 6 weeks of age and housed in a room with controlled temperature (22 ± 1 C), humidity (50-55%), and lighting (between 0800-2000 h). They were provided with food and water ad libitum. Materials An antiserum to rGRF had previously been generated in rabbits by intradermal injection of synthetic rGRF-(1-43), conjugated by glutaraldehyde to bovine thyroglobulin, and this demonstrated potent immunoneutralization of both endogenous and exogenous GRFs in male rats (17). The binding capacity of GRF antiserum calculated by Scatchard analysis of the RIA data (24) was 5.2 tig rGRF/ml serum. Synthetic rGRF was obtained from Peninsula Laboratories, Inc. (Belmont, CA), and was dissolved in physiological saline containing 0.1% BSA (Nakarai Tesque, Inc., Kyoto, Japan) just before an iv injection. Goat antimonkey r-globulin serum was purchased from Cooper Biomedical (West Chester, PA). Experimental procedure At 12-13 weeks of age, rats were implanted with a chronic Silastic (id, 0.025 in.; od, 0.047 in.; Dow-Corning, Midland, MI) intraatrial cannula, as described previously (6, 17, 25). They were then placed in individual cages, and estrous cycle and body weight were monitored daily after cannulation. Animals with at least two successive regular 4- to 5-day cycles that had fully recovered their preoperative body weight were used for experiments 2 weeks after operation. For 2 days before the experiments, the rats were accustomized to the procedures, which consisted of attachment of an extension cannulae to the atrial catheter, withdrawal of blood at 30-min intervals, and immediate reinjection without actual sampling for 3 h. Two groups of six rats, on the same day of the estrous cycle, were iv injected through the atrial catheter with either anti-rGRF antiserum (2.5 ml/kg BW) or nonimmune rabbit serum (NRS) at 1200 h. At 1600 h, both groups received an iv injection of synthetic rGRF at a dose of 1 Mg/kg BW to confirm the immunoneutralizing ability of GRF antiserum. Blood samples were collected every 15 min from 1100-1600 h and 5 and 15 min after rGRF injection. The plasma was immediately separated by centrifugation, and the red blood cells were resuspended in sterile saline and returned to the animals after the next sampling. Plasma samples were stored at -20 C until assayed. Two separate experiments were carried out, and the results were combined. GH RIA and data analysis Plasma GH was measured in duplicate by RIA (6,17) at two dilutions, using materials supplied by the Rat Pituitary Hormone Distribution Program, NIDDK, NIH. Data were expressed in terms of nanograms of rGH RP-2 per ml plasma. The tracer was added 24 h after the first antibody, and bound and free hormones were separated by goat antimonkey r-globlin serum. The minimum detectable dose was 0.9 ng/ml plasma. The intra- and interassay variations were 10.1% and 12.5%,

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respectively. GH pulses were defined as beginning when GH levels exceeded the preceding lowest GH levels over 3 coefficients of intraassay variation and to be terminated when GH values decreased from the peaks over 3 coefficients of variation. GH nadirs were defined as the lowest values preceding and/or following the GH pulses. Statistical analysis was performed by analysis of variance corrected for repeated measures, followed by the Newman-Keuls procedure or Student's t test. P < 0.05 was considered significant.

Results Figure 1 shows the secretory profiles of plasma GH and GH responses to synthetic rGRF in individual cycling female rats. Before 1200 h, when NRS or antirGRF serum was administered, pulsatile GH secretion was evident in all rats at different stages of the estrous cycle (Fig. 1, left and right panels). The NRS-treated control rats (left panel) continued to exhibit an episodic pattern of GH secretion during the following 4-h period (1200-1600 h). The secretory profiles of GH were variable within and among individual animals. GH pulses were of low amplitude (9.8-53.7 ng/ml) and occurred irregularly, with a frequency of 4-5 (mean ± SEM, 4.2 ± 0.2) surges/5 h from 1100-1600 h, whereas interpeak GH troughs were relatively high, with nadir values of 3.613.8 ng/ml. Overall mean (±SEM) GH pulses and trough nadirs were 26.8 ± 2.4 and 8.6 ± 0.7 ng/ml, respectively. All of the control rats responded to synthetic rGRF with peak GH levels 5 min after injection. Intravenous administration of GRF antiserum at 1200 h caused a dramatic suppression of both spontaneous GH secretion and the rGRF-induced rise in GH (Fig. 1, right panel). The effects occurred rapidly, within 15 min after injection, and persisted for the following 4 h. As a result, GH secretion decreased uniformly to very low levels and exhibited little or no fluctuation for the remainder of the observation period. Figure 2 shows mean (±SEM) GH levels in NRS- or GRF antiserum-treated rats. Mean GH levels were not significantly different between the two groups at 11001200 h. However, after treatment at 1200 h, mean GH levels were significantly lower in the antiserum-treated than in the control rats throughout the entire period of observation {P < 0.01, except for 1600 h where P < 0.025). GH levels decreased and were maintained at approximately 3 ng/ml from 1230-1545 h. Overall mean (±SEM) GH concentrations between 1215-1600 h were 15.3 ± 1.0 or 3.4 ± 0.1 ng/ml in the NRS- or antiserumtreated rats (P < 0.001), respectively. Synthetic rGRF induced a mean 19-fold rise in plasma GH (P < 0.001) to a peak level of 153 ± 37 ng/ml 5 min postinjection in the control rats, but did not cause a significant increase in the antiserum-treated rats. Since the GH secretory profiles were not synchronized

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GRF: PHYSIOLOGICAL ROLE IN FEMALE RATS

289 ft,

50 . p

NRS

. p Antiserum rGRF

25 •

A

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p J

50

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25

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1 1200 1400 Time of Day (h)

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of both the maximum pulses and the minimum nadirs between 1215-1600 h (bottom row). Compared with pretreatment values, the suppressive effects of GRF antiserum on both the maximum and minimum GH levels were significant for all of the fractional or entire periods. Thus, the effects of GRF antiserum consisted of suppression of both pulse and trough components of spontaneous GH secretion.

J

25

1400

1600

FIG. 2. Mean GH levels and mean GH responses to synthetic rGRF in female rats administered rGRF antiserum (•) or NRS (O). Results are expressed as the mean ± SEM of six animals. Arrows indicate the times of administration. After administration of rGRF antiserum, plasma GH levels were significantly decreased throughout the observation period. P < 0.01 vs. NRS-administered rats from 1215-1615 h, except for 1600 h where P < 0.025.

1 1

S

0 5 50

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Discussion

600

1200

1400

1600

Time of Day (h)

FIG. 1. Secretory patterns of GH and GH responses to synthetic rat GRF (rGRF; 1 /ug/kg BW) in individual conscious female rats. Right and left panels show the six individual animals treated with rGRF antiserum (2.5 ml/kg BW) or NRS, respectively. Arrows indicate the times of administration. GH levels higher than 50 ng/ml are indicated by the open circles; peak GH values after rGRF injection are indicated by the numbers beside the open circles. P, E, Di, and D2 indicate proestrus, estrus, diestrous day 1, and diestrous day 2, respectively.

among the individual female rats, the means of the lowest as well as the highest values were compared between the two treatment groups (Table 1). Comparisons were made at each of the four fractional periods from 1215-1600 h or for the entire 3.75-h period. Neither of these parameters showed a significant difference between the two groups at 1100-1200 h (Table 1, top row). Thereafter, the means of the lowest GH values as well as those of the highest values were significantly lower in the antiserum-treated rats than in the control rats at all of these fractional periods (Table 1, middle rows). Antiserum treatment also significantly suppressed the mean values

The technique of passive immunization is a powerful tool for studying the physiological role of biologically active peptides. When employed in functional studies in vivo, it is necessary to appropriately assess the extent to which an antiserum can immunoneutralize an antigenic substance. This is particularly important when the biological blocking effect of an antiserum is incomplete. While the incomplete blockade suggests at least partial involvement of a certain peptide in a specific physiological phenomenon, it does not necessarily exclude concomitant involvement of other substance(s). To investigate the immunoneutralizing capacity of GRF antiserum in vivo, we injected a supraphysiological dose of synthetic rGRF at the end of the experiments and found that the GRF antiserum could prevent synthetic GRF from causing significant stimulation of GH secretion. This effect occurred very rapidly (within 5 min) after the injection of rGRF, although the antiserum was administered as much as 4 h before rGRF injection. These results indicate that the GRF antiserum was of sufficient potency to immunoneutralize circulating GRF in female rats, which is in agreement with the previous findings obtained in male rats (7, 17). The abolishment of episodic GH secretion after GRF

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GRF: PHYSIOLOGICAL ROLE IN FEMALE RATS

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TABLE 1. Comparison of the maximum or minimum GH levels between NRS- and rGRF antiserum-treated female rats Maximum GH values Time (h)

Minimum GH values

NRS

Antiserum

P

NRS

Antiserum

P

1100-1200

31.3 ± 3.7

34.0 ± 12.1

>0.05

8.2 ± 0.8

9.0 ± 1.4

>0.05

1215-1300 1315-1400 1415-1500 1515-1600

27.9 ± 25.1 ± 29.6 ± 13.4 ±

1215-1600

37.8 ± 5.9

6.1 2.8 6.2 2.16

0.7 0.5a 0.6° 0.9a

0.005 0.001 0.005 0.005

11.1 ± 8.4 ± 8.6 ± 7.1 ±

2.0 2.1 1.3 1.1

2.8 ± 0.46 2.7 ± 0.36 2.9 ± 0.4* 2.6 ± 0.3"

0.005 0.05 0.005 0.005

5.6 ± 1.0°

0.001

5.2 ± 0.5

2.3 ± 0.46

0.001

4.3 ± 3.7 ± 4.0 ± 4.9 ±

GH values are expressed as the mean ± SEM of six rats. P < 0.025 vs. pretreatment values at 1100-1200 h. b P< 0.0025 vs. pretreatment values at 1100-1200 h.

0

a n t i s e r u m a d m i n i s t r a t i o n provides evidence t h a t G R F is

a physiological generator of spontaneous GH bursts in adult female rats. This finding substantiates the earlier observations in adult male rats that pulsatile GH secretion was inhibited by GRF antibodies (5-7) or a specific GRF antagonist (26). Thus, in spite of the striking sexual dimorphism in amplitude and frequency, GH pulses are GRF dependent in both adult male and female rats. In neonatal and young prepubertal rats of either sex, GRF also plays an important role in GH secretion. Several laboratories have reported, using a single blood determination, that passive immunization with GRF antiserum inhibits serum GH levels or decreases pituitary GH content in these rats (27-29). GRF antiserum or a peptide GRF antagonist has also been shown to suppress somatic growth or delay sexual maturation in immature rats (28, 30, 31). These findings together with the results of the earlier (5-7, 26) and present studies in adult rats support the concept that GRF is involved in postnatal development of GH secretion in both male and female rats. The absence of GH pulses after immunoneutralization of endogenous GRF suggests that other hypophysiotropic GRF-like factors, if any exist, are not involved in physiological GH secretion in rats. On the other hand, two recent studies of unanesthetized ovariectomized ewes have demonstrated that approximately 35% of jugular GH pulses are not associated with GRF pulses in the hypophysial-portal blood and suggested that these GH pulses are caused by another unknown hypothalamic factor (32) or an inherent basal activity of somatotropes (33). This discrepancy is difficult to explain, but may be due to species or methodological differences. In anesthetized male rats, portal GRF pulses parallel the times of GH secretory surges (15). By passive immunization with GRF antiserum, the secretory pattern of GRF cannot be monitored as precisely as the direct determination of portal GRF, but the former method is very useful, parhaps more suitable than the latter, for investigation of

the GRF dependency of GH secretion. The present findings provide further evidence that interpeak GH troughs as well as GH surges are dependent upon GRF. The trough GH period in the control rats often lasted over 15-30 min. If their trough GH is not maintained by GRF, it would have decreased rapidly after a short half-life in plasma (5.85 min) (34), probably to the levels observed 15-30 min after antiserum administration. Therefore, GRF in the female rat appears to be secreted during the GH trough at a level sufficient to maintain GH at a relatively high level, although we do not necessarily imply secretion of GRF at a constant rate. A decreased inhibitory tone of SRIF may also contribute to the high trough level of GH in the female rat. This is suggested by Clark and Robinson (35), who used pituitary responsiveness to repeated GRF injections as an index of endogenous SRIF secretion. However, the trough GH in the male rat is predominantly controlled by SRIF and is consistently increased by SRIF antiserum (8-14), but is not inhibited by GRF antiserum (6, 17). Collectively, these findings suggest a sexual dimorphism in the hypothalamic control of trough GH secretion. A sex-related difference has already been demonstrated in GH feedback, involving hypothalamic SRIF or GRF (36, 37), or in in vitro secretion, contents, or mRNA levels of GRF and SRIF (38-41) in the rat. Although the ultradian rhythm of GH secretion is generally accepted to result from a cyclic alternate release of GRF and SRIF in the rat (15,42), the elimination of GH secretion by GRF antiserum, but not by SRIF antiserum (8-13), suggests that GRF alone might be capable of fully generating episodic GH bursts. In fact, GRF alone can reproduce the pulsatile pattern of GH secretion in vitro when intermittently delivered to rat somatotrophs (43, 44), a pattern similar to the in vivo model of GRF secretion first proposed by Tannenbaum and Ling (42). By contrast, periodic withdrawal of SRIF infusion is not sufficient for stimulating GH secretion in the absence of GRF (43-45). Nonetheless, we do not

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GRF: PHYSIOLOGICAL ROLE IN FEMALE RATS imply that SRIF plays only a minor role in GH secretion. Within the hypothalamus, SRIF tonically inhibits GRF secretion (15-17), and when suddenly withdrawn, SRIF can induce GRF secretion in vivo (17), indicating a regulatory role of SRIF in GRF secretion. At the level of the pituitary, SRIF sensitizes somatotrophs to respond to repeated GRF pulses (44, 45) and amplifies the GRF signal when it withdraws (43-45). We suggest that when SRIF inhibits both GRF and GH secretion simultaneously at the hypothalamic and pituitary levels, GH troughs are formed, while when SRIF cyclically withdraws, disinhibiting GRF secretion and synchronously maximizing pituitary responsiveness, GH surges occur. This could be the most efficient mechanism in the presence of SRIF, by which GRF stimulates GH secretion into the bloodstream and also could explain in part why the central GH regulatory system requires SRIF in addition to GRF in the physiological regulation of GH secretion. In conclusion, the present study has demonstrated that physiological GH secretion is totally dependent upon GRF in the female rat, thereby substantiating the earlier observations in the male rat. This finding suggests that other GRF-like factors, if any exist, do not participate in physiological GH secretion in rats. Although episodic bursts of GH secretion do not occur in the absence of GRF, SRIF may play an important, probably primary, role in GH secretion by regulating both the secretion and the hypophyseal action of GRF, a generator of GH pulses.

Acknowledgment The authors are very grateful to the National Pituitary Agency, Pituitary Hormone Distribution Program, NIDDK, for kindly providing RIA kits for rat GH.

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GRF: PHYSIOLOGICAL ROLE IN FEMALE RATS

and postnatal developmental changes in hypothalamic content of rat growth hormone-releasing factor. Endocrinology 120:525-530 Arsenijevic Y, Wehrenberg WB, Conz A, Eshkol A, Sizonenko PC, Aubert ML 1988 Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor delays sexual maturation in the male rat. Endocrinology 124:3050-3059 Lumpkin MD, Mulroney SE, Haramati A 1989 Inhibition of pulsatile growth hormone (GH) secretion and somatic growth in immature rats with a synthetic GH-releasing factor antagonist. Endocrinology 124:1154-1159 Frohman LA, Downs TR, Clarke IJ, Thomas GB 1990 Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep: spontaneous secretion and response to insulin-induced hypoglycemia. J Clin Invest 86:17-24 Thomas GB, Cummins JT, Francis H, Sudbury AW, McCloud PI, Clarke IJ 1991 Effect of restricted feeding oh the relationship between hypophysial portal concentrations of growth hormone (GH)-releasing factor and somatostatin, and jugular concentrations of GH in ovariectomized ewes. Endocrinology 128:1151-1158 Badger TM, Millard WJ, Owens SM, LaRovere J, O'Sullivan D 1991 Effects of gonadal steroids on clearance of growth hormone at steady state in the rat. Endocrinology 128:1065-1072 Clark RG, Robinson ICAF 1985 Growth hormone responses to multiple injections of a fragment of human growth hormonereleasing factor in conscious male and female rats. J Endocrinol 106:281-289 Carlsson LMS, Clark RG, Robinson ICAF 1990 Sex difference in growth hormone feedback in the rat. J Endocrinol 126:27-35 Maiter DM, Gabriel SM, Koenig JI, Russell WE, Martin JB 1990 Sexual differentiation of growth hormone feedback effects on hy-

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pothalamic growth hormone-releasing hormone and somatostatin. Neuroendocrinology 51:174-180 Miki N, Ono M, Masunaga S, Shizume K, The sex difference in hypothalamic growth hormone-releasing factor (GRF) and somatostatin (SRIF) and its relation to gonadal steroids in rats. 8th International Congress of Endocrinology, Kyoto, Japan, 1988, p 98 Gabriel SM, Millard WJ, Koenig JI, Badger TM, Russell WE, Martin JB 1989 Sexual and developmental differences in peptides regulating growth hormone secretion in the rat. Neuroendocrinology 50:299-307 Chowen-Breed JA, Steiner RA, Clifton DK 1989 Sexual dimorphism and testosterone-dependent regulation of somatostatin gene expression in the periventricular nucleus of the rat brain. Endocrinology 125:357-362 Maiter D, Koenig JI, Kaplan LM 1991 Sexually dimorphic expression of the growth hormone-releasing hormone gene is not mediated by circulating gonadal hormones in the adult rat. Endocrinology 128:1709-1716 Tannenbaum GS, Ling N 1984 The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 115:19521957 Weiss J, Cronin MJ, Thorner MO 1987 Periodic interactions of GH-releasing factor and somatostatin can augment GH release in vitro. Am J Physiol 253:E508-E514 Sato M, Takahara J, Fujioka Y, Niimi M, Irino S 1988 Physiological role of growth hormone (GH)-releasing factor and somatostatin in the dynamics of GH secretion in adult male rat. Endocrinology 123:1928-1933 Soya H, Suzuki M 1988 Somatostatin rapidly restores rat growth hormone (GH) release response attenuated by prior exposure to human GH-releasing factor in vitro. Endocrinology 122:2492-2498

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Effect of antiserum to rat growth hormone (GH)-releasing factor on physiological GH secretion in the female rat.

To further study the physiological role of GH-releasing factor (GRF), we examined the effect of antiserum to rat hypothalamic GRF on spontaneous GH se...
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