Motilin stimulates growth hormone release in vitro

Brain Research Bulktin,

Vol. 8,

pp. 117-121, 1982. Printed in the U.S.A.

Motilin Stimulates Growth Hormone Release In Vitas’ WILLIS

K. SAMSON,

MICHAEL

Received

D. LUMPKIN

16 November

AND SAMUEL

M. MCCANN

1981

SAMSON, W. K., M. D. LUMPKIN AND S. M. MCCANN. Motilin stimulates growth hormone release in vitro. BRAIN RES. BULL. 8(Z) 117-121, 1382.-Motilin, a twenty two amino acid ~ly~ptide originally isolated from duodenal extracts, has been detected recently in the mammalian hypothalamus and pituitary. We have investigated the possibility that motilin might play a role in neuroendocrine events and report here the ability of synthetic porcine motilin (10V6M) to stimulate growth hormone release from rat hemipituitaries and dispersed anterior pituitary cells in vitro. No significant effects on luteinizing hormone, thyroid stimulating hormone or prolactin release were observed. Motilin may be added therefore to the growing list of gastrointestinal hormones which can act directly at the level of the anterior pituitary to alter hormone release. Motihn

Growth hormone

Growth hormone reieasing activity

MOTILIN is a polypeptide that was first purified from extracts of porcine duodenum [2] and subsequently shown to consist of twenty-two amino acids. Motilin shares some structural homology with secretin and gastrin [7]. however, the primary physiological effect of this peptide is to induce rhythmic contractions of gastric smooth muscle during interdigestive periods [4]. Synthesis of motilin (171 enabled development of antisera [22] which have been used to detect motilin in a variety of tissues. Immunoreactive motilin was first identified in gastrointestinal tissues. High levels of the peptide have also been detected in the pituitary, pineal and hy~thal~us of the dog. These findings suggested to us a possible role for the peptide in the control of anterior pituitary function, as has been described for several other gastrointestinal hormones recently detected in the CNS, such as vasoactive intestinal peptide 114,211, secretin {15], gastrin [ 191, and cholecystokinin [20]. Experiments described in this report demonstrate the in vitro growth ho~one-releasing action of synthetic motilin. METHOD

Hemipituitary

Inclubation

Paired hemipituitary incubations were conducted in medium 199 (Gibco) cont~~ng bacitracin (2x 10e5M, Sigma) and 0.1% bovine serum albumin, at 37”C, pH 7.3, in an atmosphere of 95% O,-5% COZ, as previously described [16]. Adult male rats (Sprague Dawley, Holtzman) weighing 300400 grams served as pituitary donors. Anterior pituitaries were removed following decapitation, bisected longitudinally, and placed (1 hemipituitary per tube) in 5 ml polystyrene culture tubes cont~g 2 ml incubation medium. One half of each pituitary was exposed to the appropriate treat-

ment regimen while the other half served as a paired control. After one hour of preincubation media were replaced with test media (2 ml) and the incubation continued for one hour. Upon termination of incubation, media were frozen and hemipitui~es weighed. Test media in the treatment groups consisted of 1.9 ml medium 199 plus 0.1 ml 0.9% NaCl (saline) alone or 0.1 ml saline containing test doses of motihn ranging from lo--” to 1O-8 M. Control media consisted of 2.0 ml medium 199. Data were expressed in terms of ng hormone released per mg hemipituitary and were analyzed by means of the paired t-test. Dispersed

Cell Zncubations

(Experiments

Anterior pituitaries were removed from adult male rats (Sprague Dawley, Holtzman) after decapitation and dispersed in the presence of trypsin (Difco) as previously described [16]. Cells were incubated overnight (37°C) in medium 199 containing 20 mM ~-2-hydroxye~ylpi~~~neN’-Zethanesulfonic acid (HEPES buffer, G&co), 10% horse serum and penicillin-streptomycin ( 1 ml/100 ml medium, Gibco). Cells were then pelleted on the day of experimentation and preincubation media removed. The cells were subsequently resuspended prior to incubation for two hours (3PC) in either 1 ml medium 199 (0.1% bovine serum albumin, 1% penic~~n-streptomycin, 20 mM HEPES buffer, 2 x fOm5M bacitracin) or 1 ml of the same medium containing motilin (10-6-10-8 M), somatostatin (SRIF) alone or together with motilin, or LH-releasing hormone (LRH) or thyrotropin-RI-I (TRH) as peptide controls. All peptides were obtained Tom Peninsula Laboratories. Incubations were terminated by cen~~tion (25°C 10 min, 6OOxG) and media stored frozen untii measurement of hormone content. Data were analyzed by means of Student’s f-test.

‘Supported by NIW Grants HD-09988 and AM-10073.

Copyright

@ 1982 ANKHO Intonational

3-5)

Inc.~361-9230/82/020117-05$03.~/0

118

SAMSON, LUMPKIN TABLE HORMONE

1

RELEASE (nglmg TISSUE, MEAN + SEM) FROM HEMIPITUITARIES IN VflRO (EXWRIMENT

Treatment

PAIRED 1)

MALE

RAT

PRL

TSH

LH

36.3 + 3.0

526 2 26

4.63 ? 2.39

26.3 2 6.3 10.0 2 5.5

636 2 151 110 2 171

5.69 2 1.53 1.06 f. 1.26

21.3 + 1.8

356 ?

40

1.36 X!Z 0.23

152 2 19 73.4 !z 2a.o*

17.6 + 2.7 3.7 * 2.2

564 2 75 208 -c 89

2.09 * 0.44 0.73 * 0.55

124 + 13

26.1 2 3.3

44i+

159 * 22 35.1 t 23.4

24.3 + 4.0 1.8 t 4.8

575 + 92 134 + 133

3.29 + 0.73 1.68 -+ 1.14

146 + 27

23.1 -c 3.1

504?

61

3.02 -e 0.90

181 * 32 34.3 + 61.0

27.2 + 3.2 4.2 ? 5.3

863 ” 236 359 + 296

5.04 t 1.46 2.02 t 2.08

GH

AND MCCANN

media VS

media + Saline Difference

296

t 143

298 t 131 2.7 t 49.6

media VS

media + 1O-6M Motilin Difference

79

i

12

media VS

media + lo-’ M Motilin Difference

a7

1.61 + 0.36

media VS

media + 1O-8M Motilin Difference

n=6. *p
Hormone

Assay

Luteinizing hormone (LH) concentrations of incubation media were measured by radioimmunoassay (RIA) using the RP-1 rat pituitary LH reference standard and were expressed in terms of the NIH-LH-Sl reference preparation @I. Prolactin (PRL), growth hormone (GH) and thyroid stimulating hormone (TSH) levels in incubation media were measured by the respective RIA kits provided by the NIAMDD and results expressed in terms of the RP reference standards. RESULTS

Experiments

I and 2

As previously demonstrated [ 161, saline-treated controls (mediakO.1 ml saline) failed to show any difference (Table 1 and 2) in PRL, LH, TSH or GH release when compared to the release from paired controls (media alone). Motilin, at doses ranging from 10m6to lo-* M, failed to alter significantly the release of LH, PRL or TSH from treated hemipituitaries . In both experiments, however, the 10V6M dose of motilin stimulated significantly greater @
hinder the analysis of the data since paired hemipituitaries were used as both treatment and controls and the data were analyzed by comparison of differences between pairs. Experiments

3 and 4

As in the case of hemipituitary incubations, motilin (10-6-10-* M) failed to change significantly the release of LH, TSH or PRL from dispersed anterior pituitary cells in vitro (Tables 3 and 4). That these cells could respond to peptide stimuli was demonstrated by the ability of LRH to stimulate significantly greater LH release than that seen in the presence of control media in Experiment 3 and TRH to stimulate enhanced TSH and PRL release in Experiment 4 (Tables 3 and 4). The lower doses of motilin (1O-7 and 10msM) had no significant effect on GH release from dispersed anterior pituitary cells. Once again, however, the 10es M dose of motilin stimulated significantly greater GH release than that seen from control cells. In Experiment 3, 10e6 M motilin (303.%?10.17, n=14) stimulated GH release in excess of that seen from controls (262.65k16.88, n=14), at thepc0.05 significance level. An even more pronounced stimulation (p
5

Somatostatin

significantly

inhibited

GH release

at all

MOTILIN

AND GROWTH

119

HORMONE TABLE

HORMONE

RELEASE

2

(nglmg TISSUE, MEAN f SEM) FROM PAIRED HEMIPITUITARIES IN VITRO (EXPERIMENT 2)

MALE

RAT

GH

Treatment

media

8.86 2 1.20

29

69.0 r+ 6.7

674

364 2 73.7 k

33 56.3

84.7 + 6.2 15.7 2 10.7

664 f 164 10.4 + 161.7

16.73 + 4.33 7.87 2 4.80

468

29

10.27 2 1.94

273

VS

media

+

? 136

+

Saline Difference media VS

media

2

110.7 2

7.0

536

k

88

98.3 f 12.3 k

5.0 9.0

602 2 118 65.4 2 167.2

11.20 k 2.57 0.94 2 3.19 11.23 2 1.53

+

762 2 120 294.1 5 114.2*

1O-6 M Motilin

Difference media

492

VS

media + lo-’ M Motilin Difference

+

94

111.7 + 16.6

791

? 153

676 k 183.8 _’

75 98.1

105.1 t 13.7 6.5 k 20.1

890 f 134 98.8 k 138.4

529

46

118.6 k 22.0

1040

66 64.2

105.1 + 12.1 13.6 2 20.6

1121 2 350 80.9 f 273.5

19.90 f 2.66 8.66 f 3.17

media VS

media + lo-” M Motilin Difference

+

457 k 72.8 k

14.42 k 2.49

2508

23.19 + 8.15 8.77 f 8.99

n=6. *p
doses tested (Table 5). This inhibition was maximal already at the lo-’ M dose level. As in the two previous experiments, 10ms M motilin stimulated significantly greater GH release @
release. Indeed the inhibitions of GH release seen with all three doses of SRIF were not significantly altered when motilin was present. DISCUSSION

A new pattern seems to be emerging in the field of neuro-

TABLE 4

TABLE 3 HORMONE RELEASE

MEAN + SEM) FROM CELLS DISPERSED FROM ADULT MALE RAT PITUITARIES (EXPERIMENT 4)

HORMONE RELEASE (ng/lW CELLS, MEAN ? SEM) FROM CELLS DISPERSED FROM ADULT MALE RAT PITUITARIES (EXPERIMENT 3)

Treatment

n

GH

PRL

LH

TSH

Control

14

263 217

20.2 20.7

2.25 20.13

(q/l@ CELLS,

Treatment

n

GH

192 +ll

Control

14

382 224

PRL

LH

TSH

10.8 kl.3

0.74 kO.11

120 +17

lO-B M Motilin

14

304* 210

20.1 k1.0

2.38 20.12

174 k12

lO-B M Motilin

14

514t 231

11.1 kl.3

0.73 kO.08

110 58

IO-’ M Motilin

14

268 k7

20.1 kO.7

2.38 kO.12

157 28

lo-’ M Motilin

14

438 224

10.1 kO.9

0.81 20.11

120 +11

lO-8 M Motilin

13

290 213

18.5 21.3

2.24 20.06

186 k6

1O-8 M Motilin

10

424 *23

11.3 21.3

0.74 kO.04

106 +8

lo-’ M LHRH

3

lo+ M TRH

4

5.42t kO.30

*Versus controlp
control

p<
*Versus control pCO.05. tVersus control p
16.6* a2.2

4583: 239

120

SAMSON, TABLE

5

EFFECTS OF MOTILIN AND SOMATOSTATIN (SRIF) ON GROWTH HORMONE RELEASE fngll0’ CELLS, MEAN rt SEM) FROM CELLS DISPERSED FROM ADULT MALE RAT PITUITARIES (EXPERIMENT 5)

Treatment

GH Released

Control lo+ M SRIF lo-’ M SRIF lo-” M SRIF 10e6 M Motilin 10m7M Motilin lO-6 M SRIF + lO-6 M Motilin lo-’ M SRIF + lO-6 M Motilin lo-* M SRIF + lOmBM Motilin

289 5 18 178 2 II* 171 f 6* 232 ? 7* 386 t 14* 307 -+ 26 176 + 13* 1.56 2

8*

231 t 19*

ll=7.

*p
endocrinology. Peptide hormones previously thought to be solely of gut origin now are being discovered in a variety of neural tissues [9, 10, 12, 14, 221, as well as in the pituitary of several species. The presence of these peptides in CNS structures known to be important in the control of anterior pituitary function strongly suggests a role for them in hypothalamic-pituitary interactions [6]. Indeed, the ability of four such hormones, known to be present in the brain or pituitary, VIP [21], secretin [15], gastrin [19] and cholecystokinin [20], to affect anterior pituitary hormone secretion has been described. With this in mind, the demonstration of immunoassayable motilin in the mammalian pituitary and hypothalamus [9,22], including especially the median eminence [9], stimulated our examination of the possible direct action of this peptide on anterior pituitary hormone release. In both hemipituitary (Tables 1 and 2) and dispersed cell (Tables 3-5) incubations, motilin (10e6 W) was capable of stimulating significantly greater GH release than that seen in the presence of control media. The pharmacologic specificity of this effect is suggested by the inability of this dose of motilin to alter the release of LH, TSH or PRL. In the case of hemipituitary incubations, sufficient incubation media remained after assay of these hormones to measure follicle stimulating hormone (FSH) release by RIA. No significant effect of motilin

LUMPKIN

AND MCCANN

on FSH release was observed (data not shown). Additionally, the inability of the effective GH-releasing dose ( 10e6 M) of motilin to overcome SRIF-induced inhibition of GH release (Experiment 5) suggests that the action of motilin to release GH is expressed through receptors other than those for SRIF and that this stimulatory action of motilin can be overridden by the inhibitory effect of SRIF. The failure of lower doses of motilin to alter GH release prevents any assumption of the physiologic role of motilin in this event. The effective dose, lo-” M, is in excess of circulating motilin levels reported in other mammals [4]. This is also, however, the case with VIP’s ability to stimulate PRL release in vitro [16] where levels of the peptide higher than those found in peripheral plasma [13] were required. It is also possible that, as in the case of VIP [13], levels of motilin in the hypophysial portal plasma greatly exceed those found in the peripheral circulation, suggesting a more physiologic range of the effective dosage. Additionally, it should be noted that other gastrointestinal peptides, including VIP [16], secretin [15] and cholecystokinin (W. K. Samson, J. I. Koenig and S. M. McCann, manuscript in preparation), when tested under similar in vitro conditions failed, at even higher doses than that required for motilin, to alter GH release. This fact again argues in favor of the pharmacologic specificity of motilin’s GH-releasing action, even at lOAfiM doses. On the other hand, since the synthetic motilin is the porcine equivalent, it is possible that some subtle difference in structure occurs across species and that rat motilin, if indeed slightly different in structure, would be more biologically active. Heterogeneous forms of immunoreactive motilin have been described [18] and it has been suggested [lo] that rat secretin differs somewhat structurally from the porcine molecule. Until now little has been reported concerning the neuroendocrine role of motilin. It is known that circulating levels of the peptide are elevated during phase III of interdigestive myoelectric activity in the duodenum [4] and it is thought that this elevation mirrors its role in digestive processes. What evidence exists for a relationship between the neuroendocrine control of growth hormone secretion and motilin? Late postprandial elevations in GH have been described and the stimuli for them have not been agreed upon [ 1,111. These elevations appear to coincide with the interdigestive phase during which motilin is elevated [4]. Additionally, plasma motilin concentrations are elevated in some patients with diabetes mellitus [3], a condition often accompanied by elevated GH levels [5]. These observations suggest a possible role for motilin in GH secretion by a direct action on the pituitary to stimulate GH release. Motilin can be added, therefore, to the list of gastrointestinal hormones recently localized to the CNS for which a neuroendocrine role may be implied.

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AND GROWTH

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