0013.7227/92/1305-2579$03.00/O Endocrinology Copyright 0 1992 by The Endocrine

Vol. Printed

Society

Role of Selected Endogenous Peptides in Growth Hormone-Releasing Hexapeptide Activity: Analysis Growth Hormone-Releasing Hormone, Thyroid Hormone-Releasing Hormone, and GonadotropinReleasing Hormone* BARRY

B. BERCU,

SEI-WON

Department of Pediatrics, University St. Petersburg, Florida 33701

YANG, of

RYUJI

South Florida,

MASUDA,

AND

Tampa, Florida

ABSTRACT.

The purpose of this study was to evaluate the contribution of endogenous GH-releasing-hormone (GHRH) to exoaenous GH-releasina hexapeptide (GHRP-6) activitv. and to determine whether TRi-I or GnRH are endogenous analogs of GHRP-6. The activity of GHRP-6, a synthetic GH secretagogue, was significantly attenuated in rats administered GHRH antiserum or a-methyl-p-tyrosine to reduce endogenous GHRH concentrations, andalso in rats administered 5-50 rg/kg of [N-AcTvrl. D-Are’?-GRF 1-29 amide to block pituitarv GHRH recento”rs.‘Howe&, GHRP-6 activity was poientiated in rats administered 150 rg/kg [N-Ac-Tyr’, D-Arg*]-GRF 1-29 amide, presumably due to partial agonist activity of the GHRH receptor antagonist at the higher dose. These data show that endogenous GHRH contributes to full expression of exogenous GHRP-6

RICHARD

130, No. 5 in U.S.A.

of

F. WALKER

33612; and All Children’s

Hospital,

activity in vivo. Like TRH, a subthreshold dose of GHRP-6 was significantly more effective in hypothyroid rats than in euthyroid rats. However, suprathreshold doses of GHRP-6 were less effective in hypothyroid rats. Unlike TRH, GHRP-6 had no effect on GH and prolactin release from GH, cells, and TRH and GnRH were poor competitors for %GHRP-6 binding sites on pituitary membranes. A GnRH receptor antagonist did not block GHRP6 activity in vivo, and GnRH administered alone or in combination with GHRP-6, did not stimulate GH release. The results of this study suggest that synergy between GHRH and GHRP-6 seen in pharmacological studies is physiologically relevant, and that TRH and GnRH are not endogenous analogs of GHRP-6. (Endocrinology 130: 2579-2586, 1992)

G

H-releasing hexapeptide, GHRP-G(His-D-Trp-AlaTrp-D-Phe-Lys-NH& is a synthetic molecule that stimulates GH release in all species tested (1). Slight interspecies differences in potency have been observed with primates, especially man, being most sensitive to GHRP-6 (2-4). Several studies have shown that GHRP6 potentiates GH-releasing hormone (GHRH) activity when the peptides are coadministered (5-7). However, the physiological relevance of GHRP-G/GHRH synergy has received relatively little attention, and there is still some question as to whether endogenous GHRH actually contributes to GHRP-6 activity in uiuo. GHRP-6 is probably not a synthetic analog of GHRH since the two peptides employ different binding sites (B), different intracellular second messengers (5) and produce different patterns of GH secretion (9). Instead, GHRP-

6 may complement GHRH activity by mimicking the activity of an unidentified endogenous substance. Unique GHRP-6 binding sites on pituitary membranes that are quantitatively and qualitatively different from GHRH binding sites support this hypothesis (8). Many peptides release GH under pathological or experimental conditions. Some peptides such as vasoactive intestinal peptide have binding site and second messenger characteristics in common with GHRH (10) and, thus, can be excluded from consideration as endogenous GHRP-6 analogs. Other peptides such as TRH and GnRH are physiologically relevant modulators of GH secretion (ll12), with functional characteristics different from GHRH. A paracrine role for these peptides has been proposed in which they modify GHRH-mediated GH release at a local pituitary level by affecting intercellular communication (13). Although GHRP-6, GnRH, and TRH are not structural homologs, certain functional similarities suggest that one of the endogenous peptides may be a GHRP-6 analog. A functional link between the peptides derives from the fact that GHRH potentiates

Received November 18,1991. Address all correspondence and requests for reprints to: Dr. Richard F. Walker. Division of Endocrinoloev. Denartment of Pediatrics. All Children’s’ Hospital, 801 Sixth Street’ South, St. Petersburg, Florida 33731-8920. * Supported in part by Ares-Serono Group.

2579

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2580

ENDOGENOUS

PEPTIDES

the GH-releasing activities of TRH (14, 15), GnRH (16, 17), and GHRP-6 (5, 7). Furthermore, K-opioid receptors in the pituitary affect the GH-releasing activity of GnRH and dynorphin-related peptides (K-opioids) have been identified as potent competitors for GHRP-6 pituitary binding sites (18). Thus, GHRP-6 may be a synthetic superagonist for pituitary loci used by TRH or GnRH to release GH under certain pathological or experimental conditions. Based upon these relationships, the present study was designed to investigate the functional interaction between GHRP-6 and endogenous GHRH, and to determine whether TRH and/or GnRH represent naturally occurring analogs of GHRP-6.

Materials Animal

and Methods

husbandry

Sexually mature (90-120 day old) female Sprague-Dawley rats (Charles River Breeding Labs; Wilmington, NC) weighing between290-340g wereusedin this study after at least 2 weeks acclimation to local housing conditions. Female rats were the modelchosenfor study becausethey respondmore consistently and reproducibly to GHRP-6 than do consciousmales (19). Housing conditions included alternating 12-h periods of light and darkness, 60 + 5% relative humidity, food (Purina Rat Chow; Purina Ralston Company, Kalamazoo, MI) and tap water ad libitum. The animal facility in which this study was performed is fully approved for use by the AAALAC and complieswith federal statutes and regulations relating to animal usein research. Blood sampling

Samples for analysis of plasma GH concentrations were collected between 0900 and 1100 h, except when surgical proceduresand sequential samplingconsumeda full day (-08001800h). When collection intervals were extended, sampleswere drawn alternatively from the different groups so as to control for time of day variation. Single point determination of GHRP-6 activity in conscious female rats wasaccomplishedby administeringthe hexapeptide by SCinjection. Fifteen minutes later, the rats were decapitated with a guillotine, and trunk blood was collected in heparinized centrifuge tubes for subsequentanalysis of plasmaGH concentrations. Rats awaiting decapitation were isolated in an adjacent room to avoid potential stressresulting from soundsand smellsassociatedwith the procedure. When administered SC, GHRP-6 is rapidly absorbedand stimulates GH releasein a manner that is indistinguishablefrom that following iv administration (19), precluding the need to restrain the rats for iv administration via the tail. These characteristics allow GHRP6 administration with minimal stress, and support the use of SCadministration wheneverpossible.When specific experimental paradigmsrequired lessstable and/or morepoorly absorbed compoundssuch as GHRH, GHRH antiserum, and GHRH/ GnRH receptor antagonists to be administered to conscious, restrained rats via their tail veins, GHRP-6 was also adminis-

AND

GHRP-6

Endo. Voll30.

1992 No 5

tered iv. Multiple point analyses of GH secretion in anesthetized femalerats were accomplishedvia cannulaethat weresurgically placed within the femoral artery/vein after administration of ketamine (50 mg/kg, im) and pentobarbital (35 mg/kg, ip). All rats in a given experiment were surgically prepared en masse, and different treatments were alternated between individual animals so that anesthesiatimes for all groups were approximately the same.Solutions of peptides (1 ml/kg) were administered as 30 set intraarterial infusions, and blood samplesfor GH analysiswerecollected at designatedtimes into heparinized tubes. Plasma GH concentrations in these sampleswere estimated by RIA. Hypothyroidism

Methimazole, an inhibitor of iodine organification in the thyroid, wasaddedto the rats drinking water at a concentration of 0.1 mg/ml and provided ad lib&m for 14 consecutive days. Hypothyroidism in methimazole-treated rats wasconfirmed at necropsy by measuringplasmathyroxine and triiodothyronine concentrations and comparing the values to those derived from euthyroid controls. Cell culture

GH, cells (rat pituitary tumor, APCC CCL 82.1; American Tissue-Type Culture Collection, Rockville, MD 20852)usedto compare the effects of GHRP-6 and TRH on GH and PRL releasewere maintained in Ham’s FlO medium supplemented with 15%horseserum,2.5% fetal calf serum,50U/ml penicillin, 50 rg/ml streptomycin, and 1.25 pg/ml amphotericin B. Cultures were plated at concentrations of 5 x lo4 cellsper well (24 well plates) and incubated at 37 C in a humidified atmosphere of 5% CO, and 95% air. Each treatment (various dosesof GHRP-6 or TRH) was tested in six wells. Immediately after exposure to GHRP-6 or TRH, medium from each well was removed, centrifuged, and the supernatant frozen for subsequent measurementof GH and PRL concentrations. Passive immunization

against GHRH

GHRH antiserum kindly provided by Dr. W. B. Wehrenberg (20, 21), was administered iv, 2 h before evaluation of GHRP6 activity. Blood sampleswere subsequently collected as describedabove for determination of GH concentrations. Binding

sites

TRH and GnRH were tested for competition with GHRP-6 binding sites by incubating various concentrations ( 1O-g-1O-4 M) of the endogenouspeptides with approximately 1 nM “HGHRP-6. The binding assay was performed using pituitary membranesas previously described(8). Chemicals

GHRP-6, [N-Ac-Tyr’, D-Arg*]-GRFl-29 amide, [D-Phe,, Pro3, D-Phe’]-LH-RH, TRH, GnRH, and methimazole, CXmethyl-p-tyrosine were purchasedfrom PeninsulaLaboratories (Belmont, CA) and SigmaChemical Company (St. Louis, MO), respectively. Controls received saline administered in equal volume and by the sameroute in each separateexperiment.

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ENDOGENOUS Quuntitation

of

hormone

PEPTIDES

concentrations

GH concentrations in plasma (20 ~1) and GH and PRL concentrationsin incubation mediafrom GH, cell cultures were estimated in duplicate by RIA and expressedin terms of the reference preparations rGH-RP-2 and rPRL-RP-3, respectively, from the NIDDK. Radiolabeledrat GH and PRL were purchasedfrom Chemicon International, Inc. (Temecula, CA) and precipitating antibody (goat antimonkey y y, P-4) was purchased from Antibodies Inc. (Davis, CA) and used at a concentration of 1:lO. Intraassay and interassaycoefficients of variation were lessthan 5 and 8%, respectively. PlasmaT:, and Tq in methimazole-treated and control rats were estimated using commercially available RIA kits (Diagnostic Products, Los Angeles,CA). Statistical

analysis

Differences in singletime-point, plasma GH, or culture media concentrations were analyzed using Student’s t test. Temporal GH secretory profiles were performed using analysis of variance and repeated measuresanalysis. A p value lessthan 0.05 was used to define statistically significant differences. Competitive binding data for TRH and GnRH vs. “H-GHRP6 wereanalyzed usingthe computer program Lundon (Lundon Software, Cleveland, OH). The binding parameters previously determined for “H-GHRP-6 (8) were used to enable the computer to perform nonlinear modeling of each potential inhibitor’s dose-response profile.

Results The purpose of the first experiment was to assessthe contribution of endogenous GHRH to GHRP-6 activity. Stimulated GH secretion was compared in rats preadministered vehicle (controls) or one of two substances used to lower endogenous GHRH concentrations. One of the treated groups was administered GHRH antiserum

240

/J

h

2

A

T

200

c 2160

1

3

100 ;;OHRP-6

;g,lcg

300

1000

S.C.)

1. Doseresponse curvefor GHRP-6 in ratspretreatedwith vehicle (triangles), a-methyl-p-tyrosine (circles), or GHRH antiserum

FIG.

(squares). Values represent mean + SEM (8 rats per dose and treatment) in plasma collected 15 min after administration of GHRP-6.

AND

GHRP-6

2581

to passively immunize against circulating GHRH (20, 21). The other group was administered a-methyl-p-tyrosine (100 mg/kg SC x 4 days) an inhibitor of norepinephrine synthesis, to reduce adrenergic stimulation of hypothalamic neurosecretory neurons and, thus, partially lower GHRH concentrations available to the pituitary. As seen in Fig. 1, amplitude of the dose-response curve for GHRP-6 in conscious female rats was reduced approximately 90% after passive immunization against GHRH and approximately 70% after pretreatment with c-u-methyl-p-tyrosine. The second experiment was designed to determine whether a pituitary membrane antagonist of GHRH receptors (22) altered GHRP-6 activity in uiuo. [N-AcTyr’, D-Arg*]-GRF l-29 amide was administered to block access of endogenous GHRH to its pituitary receptor, and 5 min later a bolus of GHRP-6 (30 pg/kg, intraarterial) was administered. A separate group of rats was administered morphine (1.5 mg/kg) instead of GHRP-6, since the opioid causes GH secretion by releasing GHRH (21). As seen in Fig. 2, GHRP-6-mediated GH release was suppressed in a dose-related fashion by pretreatment with 5-50 pg/kg [N-Ac-Tyr’, D-Arg’]-GRF l-29 amide (P < 0.05). However, GHRP-6 activity was potentiated in rats pretreated with 150 pg/kg [N-Ac-Tyr’, D-A&]GRF l-29 amide, elevating plasma GH concentrations nearly 5-fold above control values (P < 0.01). In contrast, morphine-stimulated GH secretion was suppressed at all doses of [N-Ac-Tyr’, D-Arg2]-GRF l-29 amide (statistical significance reached at 50 and 150 mg/kg GHRH antagonist). The data presented in Table 1 show that plasma GH concentrations were significantly elevated in rats administered 150 pg/kg [N-Ac-Tyr’, D-Arg2]-GRF l-29 amide alone, suggesting that at this high dose, the molecule expressed characteristics of a GHRH receptor partial agonist. Thus, nominal activation of GHRH receptors profoundly potentiated GHRP-6 activity while having little effect on morphine-stimulated GH release. In the next study, functional similarities between GHRP-6, TRH, and GnRH were examined. The purpose of first experiment was to assessthe activity of GHRP6 in hypothyroidism, a pathological condition in which the GH-releasing potential of TRH is expressed (see Ref. 11). Hypothyroidism was produced in one group of rats by exposure to methimazole (0.1 mg/ml) in their drinking water. As seen in Table 2, plasma T3 and T4 concentrations were 63 and 96% lower, respectively, in methimazole-treated rats compared to control rats. Based upon the dose-response in conscious female rats previously described, the effects of 10 pg/kg (subthreshold) and 100 pg/kg (ED& GHRP-6 were compared in hypothyroid and euthyroid animals. As seen in Fig. 3, hypothyroid rats released significantly more GH (P c 0.01) than euthyroid rats in response to the subthreshold dose of

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ENDOGENOUS

2582

PEPTIDES

AND

GHRP-6

Endo. Vol130.No5 600

1300

6

A 1200

FIG. 2. Effect of [N-Ac-Tyr’, D-A$]GRF 1-29 amide, a GH receptor antagonist, on GH release in response to GHRP-6 (30 rg/kg, closed symbols, panel A) or morphine (1.5 mg/kg, open symbols, panel B). The GHRH receptor antagonist was administered in doses of 0 (circle), 5 (square), 50 (triangle), or 150 (diamond) rg/kg. Values represent mean + SEM 8 rats per group. GHRP-6 stimulated GH secretion as a function of time was significantly reduced (P c 0.05) by 50 mg/kg GHRH receptor antagonist and significantly increased (P < 0.01) by 150 mg/kg GHRH receptor antagonist. Morphine-stimulated GH secretion was significantly reduced (P < 0.05) by 50 and 150 mg/kg GHRH receptor antagonist.

11W

m

low

/7 5co

400 3 E 3400 G

3w

5 % uJm 2

2w 200

loo loo

0

0

0

5

10

TIME

TABLE 1. Effect of [N-Ac-Tyr’, D-A$]-GRF l-29 amide, a GH receptor antagonist, on basal, plasma GH concentrations in female rats

Dose of GH antagonist (rglk) 0

Plasma basal GH concentrations (ndml) 5.7 + 0.9 6.9 3~ 1.1 4.8 + 0.8 18.7 + 2.3”

5 50 150

Values represent mean + SEM of GH concentrations collected 20 min after administration of the GH receptor antagonist (n = 6 rats per dose). ’ P < 0.01 compared to all other values. TABLE 2. Plasma concentrations of thyroid hormones in female rats administered methimazole (0.1 mg/ml) in drinking water for 14 consecutive days

Treatment

Triiodothyronine (T3; nddl)

Vehicle 72.83 Methimazole 27.25 Values represent mean f SEM rats per group. ’ P < 0.01 compared to vehicle

+ 3.10 + 1.78

Thyroxine V4; r&N 2.84 + 0.71

0.11 + 0.04” for duplicate determinations from 7 treated (controls).

GHRP-6, whereas they released less GH (P < 0.001) than euthyroid rats in response to the EDS0 dose. Although the difference in GH secretion between euthyroid and hypothyroid rats administered the subthreshold dose of GHRP-6 was statistically significant, it represented a relatively small change in absolute concentrations of plasma GH (approximately 10 rig/ml). The secretory dynamics of GHRP-6 were then studied in anesthetized

15

(minutes)

30

0

5

10

TIME

1.5

(minutes)

rats. This model has been previously shown to be appropriate for acute, temporal analysis of GHRP-6 mediated GH release (3). The low end of the dose-response curve was evaluated to determine the extent to which suprathreshold doses of GHRP-6 are enhanced by hypothyroidism. Dose selection was based upon additional doseresponse data gathered in the anesthetized female rat (Fig. 4A). The data presented in Fig. 4B show that unlike the subthreshold dose, suprathreshold doses of GHRP-6 released significantly less GH in hypothyroid rats than in euthyroid rats. Thus, the enhanced sensitivity to GHRP-6 in hypothyroid rats could only be detected at a dose that did not change basal GH concentrations in euthyroid rats. The purpose of the next experiment was to determine if GHRP-6 like TRH, stimulates the release of GH and/ or PRL from GH, tumor cells (23). As seen in Table 3, GHRP-6 had no effect on GH or PRL release, unlike TRH which stimulated the secretion of both pituitary hormones. The ability of TRH and GnRH to compete for GHRP6 binding sites on pituitary membranes was then tested. As seen in Fig. 5, TRH and GnRH were poor competitors for 3H-GHRP-6 binding, producing only about 10% displacement at micromolar concentrations. The purpose of the next study was to determine whether preadministration of a GnRH receptor antagonist (24) blocked GHRP-6 activity. The data presented in Table 4 show that GHRP-g-mediated GH release was not significantly altered by preadministration of [D-Phe2, Pro3, D-Phe’]-LH-RH over a range of doses (5-100 pg/

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ENDOGENOUS

PEPTIDES

AND

GHRP-6

2583

3ml A

260

B

260 240

FIG. 3. Effect of methimizole-induced hypothyroidism on plasma GH concentrations in conscious female rats administered a subthreshold (10 rg/kg, A) or suprathreshold (100 rg/kg, B) dose of GHRP-6. Each symbol represents the value of an individual rat. Horizontal lines represent the means and vertical lines represent the SEM for each group.

4%

-F 8

60

n

I

EUTHYROID

HYPOTHYROID

EUTHYROID

I

HYPOTHYROID

1

A

FIG. 4. GHRP-6 dose response curve (euthyroid rats, A) and GH secretory profiles for GHRP-6 (B) in hypothyroid (open symbols) or euthyroid (closed symbols), anesthetized, female rats. Doses of GHRP-6 presented in panel B are 6 pg/ kg (circles), 3 rg/kg (squares), and 0 rg/ kg (triangles). Values represent means + SEM for six rats per dose or treatment group. GH secretion as a function of time was significantly greater (P < 0.05) in both euthyroid groups than in hypothyroid groups.

60 -

o’,,

, 0.1 0.3

0.6

I 1

DOSE

I 3

GHRP-6

kg), and that GnRH was an ineffective GH secretagogue when administered alone or in combination with GHRP6. Discussion The results of this study show that potentiation of coadministered GHRH and GHRP-6 is physiologically

@g/kg)

0

‘, 0

I 5

I 10

I 15

TIME

(minutes)

I 20

I 30

relevant, and that TRH or GnRH are not functional, endogenous analogs of GHRP-6. Previous studies, both in vitro (5, 6) and in uiuo (1, 7), have demonstrated potentiation by co-administered GHRP-6 and GHRH, such that stimulated GH release far exceeds the sum of GH release produced by administration of either peptide alone. However, the physiological relevance of this interaction has not been established. Since the concentrations

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ENDOGENOUS

2584 TABLE

PEPTIDES

3. GH and PRL secretion from GH3 cells exposed to GHRP-6

or TRH Treatment None (control) TRH (50 nM) GHRP-6 (1 nM) GHRP-6 (3 nM) GHRP-6 (10 nM) GHRP-6 (30 nM) GHRP-6 (100 nM) GHRP-6 (300 nM) GHRP-6 (1 wM)

Growth hormone concentration (ng) 2.06 f 0.44 5.31 2 0.87” 1.85 f 0.50 2.19 + 0.33 2.15 f 0.62 2.36 + 0.47 2.05 + 0.19 2.28 + 0.21 2.18 + 0.44

Prolactin concentration (ng) 0.45 f 0.03 1.04 f 0.09” 0.48 + 0.06 0.39 + 0.05 0.38 + 0.02 0.41 + 0.03 0.50 + 0.03 0.53 f 0.06 0.58 f 0.05

Values represent mean + SEM hormone concentrations released by 5 x lo4 cells/100 ~1 incubation media per 30 min. “P < 0.01 compared to unstimulated or GHRP-6 stimulated (all doses).

of exogenous GHRH used in prior potentiation studies exceeded those occurring naturally in the pituitary circulation, especially during low-level spontaneous GH secretion, the findings had little relevance to endogenous GHRH-enhanced GHRP-6 activity. In the present study, passive immunization against endogenous GHRH produced nearly total suppression of GHRP-6 activity. Possible interpretations of these data are that GHRP-6 activity requires at least minimal activation of pituitary GHRH receptors, or that GHRP-6 stimulates GHRH release in a dose-related fashion. To test these hypotheses, a group of rats was administered [N-Ac-Tyr’, D-Arg’]-GRF l-29 amide, a GHRH-receptor antagonist (22) before receiving GHRP-6 or morphine. Morphine loo

80

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Endo. 1992 Vol 130. No 5

GHRP-6

and GHRP-6 were compared because the GH-releasing activity of the opioid is directly related to endogenous GHRH release (21). The differential responses of GHRP-6 and morphine to high doses of [N-Ac-Tyr’, DArti]-GRF l-29 amide suggested that release of endogenous GHRH plays only a small part, if any, in the mechanism of GHRP-6 action. This hypothesis is based upon the fact that low doses of the antagonist which are devoid of partial agonist activity, had comparable effects on GHRP-6 and morphine activity. However, at the high dose when partial agonist activity of [N-Ac-Tyr’, D-A&] -GRF l-29 amide was expressed, morphine activity was reduced below control values and baseline GH values were only slightly elevated. In contrast, GHRP-6 activity was markedly stimulated, producing plasma GH levels that significantly exceeded the range observed when GHRP-6 was administered alone. Considering the magnitude of the potentiation and the relatively low GHRH activity expressed at the high dose of the GHRH antagonist, it seems that the physiological concentrations needed to express GHRP-6 activity, when administered alone, are exceedingly low. Thus, GHRP-6-mediated GH release in vivo is related quantitatively to endogenous GHRH concentrations. The data suggest a physiological dependence upon GHRH receptor occupancy for GHRP6 expression in vivo and, also, that the activity of GHRH may, in turn, reflect the presence of a yet unidentified endogenous analog of GHRP-6. The GH-releasing activity of GHRP-6 was compared with TRH and GnRH to determine if either of these naturally occurring molecules represents an endogenous

-

-

is s iii T

FIG. 5. Inhibition of “H-GHRP-6 binding to pituitary membranes by various concentrations of GHRP-6 (circles), TRH (triangles), or GnRH (squares).

0 60-

E 3

40-

6 E

B 8

20-

9

8

7

6

5

PEPTIDE CONCENTRATION (-log M)

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4

ENDOGENOUS

PEPTIDES

TABLE 4. Effect of GnRH antagonist (100 pg/kg) or GnRH (100 rg/ kg), administered alone or in combination with GHRP-6 (100 @g/kg) on plasma GH concentrations in female rats Treatment Saline GHRP-6 GHRP-6 + GnRH GHRP-6 + GnRH GnRH antagonist GnRH

Plasma

antagonist

GH

5.1 123 144 107 7.3 5.8

+ + + t + +

(rig/ml) 0.6 18.7 23.0 20.7 1.0 0.8

Values represent mean & SEM (6 rats per treatment) hormone concentrations in plasma collected 15 min after peptide administration.

GHRP-6 analog. Although the GH releasing potentials of TRH and GnRH are expressed only under pathological or experimental conditions, GHRP-6 could represent a superagonist for their GH-releasing binding sites and, thus, be expressed under normal conditions. In fact, the activity of GHRP-6 in models devoid of GHRH or its receptors is low or even absent (25). Since the GHreleasing potential of TRH is expressed by GHRH (14), a link between TRH and GHRP-6 was suggested. Hypothyroidism, a condition in which TRH releases GH, was used to compare TRH and GHRP-6 activity. If GHRP-6 released GH through a TRH-like mechanism, the hexapeptide’s activity should have been enhanced in hypothyroid rats. Initially, GHRP-6 was active at a dose which was subthreshold in euthyroid rats, thus supporting the hypothesis that TRH is an endogenous analog of GHRP-6. However, at higher doses, hypothyroidism attenuated GHRP-6 activity as previously reported (26). The paradoxical difference in response at subthreshold and suprathreshold doses can be explained by the fact that GHRH release is enhanced (27), while pituitary GH synthesis and content are reduced in hypothyroidism. Thus, at the low dose of GHRP-6, elevated concentrations of endogenous GHRH probably amplified GHRP6 activity causing plasma GH levels to significantly increase above baseline. However, the absolute increase in plasma GH was small (~15 rig/ml). Thus, stimulated GH secretion in hypothyroid rats may have exceeded that in euthyroid rats because at the very low dose of GHRP-6, GH release represented only a small fraction of the releasable pituitary GH pool. However, when a higher dose of GHRP-6 was administered, the reduced pituitary GH pool in hypothyroid rats prevented full expression of GHRP-6 activity despite the elevated levels of endogenous GHRH. It is presently unclear whether similar potentiative interactions exist for TRH and endogenous GHRH in hypothyroid and euthyroid rats because the GH-releasing potential of TRH is not expressed under euthyroid conditions. In fact, elevated concentrations of endogenous GHRH may be a (the) factor responsible for TRH-mediated GH secretion in hypothyroidism. GH, cells were used to further investigate whether

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GHRP-6

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TRH is an endogenous analog of GHRP-6 since TRH, but not GHRH, released GH from these cells (23,28). In the present study, GHRP-6 had no effect on GH or PRL release suggesting a mechanism different from TRH. Finally, when TRH and GnRH were coincubated with 3H-GHRP-6 they were poor competitors for the hexapeptide binding sites on pituitary membranes. GnRH was examined as a potential endogenous analog of GHRP-6 because it also stimulates GH secretion when endogenous GHRH concentrations are elevated (16, 17), and because GnRH is a GH-releasing hormone in lower vertebrate species (12). The GnRH antagonist [D-Phe,, Pro3, D-Phe’]LH-RH was preadministered to rats in an attempt to reduce GHRP-6 activity. However, GHRP-6mediated GH release was not changed over a range of [D-Phez, Pro3, D-Phe’]LH-RH doses. Furthermore, the GH-releasing potential of GnRH was not expressed in normal female rats, when the peptide was administered alone or with GHRP-6. In conclusion, the results of this study show that endogenous GHRH is physiologically relevant to expression of GHRP-6 activity in U~‘UO,and that TRH or GnRH are probably not endogenous analogs of the hexapeptide. References 1. Bowers CY, Momany FA, Reynolds GA, Hong A 1984 On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology 114:1537-1545 2. Bowers CY, Sartor AO, Reynolds GA, Badger TM 1991 On the actions of the growth hormone-releasing hexapeptide, GHRP. En- docrinology 128:2027-2035 3. Walker RF. Codd EE. Barone FC. Nelson AH. Goodwin T. Camobell SA 1991 Oral activity of the growth hormone releasing~peptihe His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in rats, dogs, and monkeys. Life Sci 47:29-36 4. Ilson BE, Jorkasky DK, Curnow TR, Stote RM 1989 Effect of a new synthetic hexapeptide to selectively stimulate growth hormone release in healthy human subjects. J Clin Endocrinol Metab 69:212-214 5. Cheng K, Chan WW-S, Barreta A, Convey DM, Smith RG 1989 The synergistic effects of His-D-Trp-Ala-TrpD-Phe-Lys-NH, on growth hormone (GH)-releasing factor-stimulated GH release and intracellular adenosine 3’,5’-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology 124:2791-2798 6. Blake AD. Smith RG 1991 Desensitization studies usina nerifused pituitary cells show that growth hormone-releasing ho&rone and His-D-Trp-Ala-Trp-D-Phe-Lys-NH? stimulate growth hormone release through distinct receptor sites. J Endocrinol 129:11-19 7. Bowers CY, Reynolds GH, Durham D, Barrera CM, Pezzoli SS, Thorner MO 1990 Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GHreleasing hormone. J Clin Endocrinol Metab 70:975-982 8. Codd EE, Shu AYL, Walker RF 1989 Binding of a growth hormone releasing hexapeptide to specific hypothalamic and pituitary binding sites. Neuropharmacology 281139-1144 9. McCormick GF, Millard WJ, Badger TM, Bowers CY, Martin JB 1985 Dose-response characteristics of various peptides with growth hormone-releasing activity in the unanesthetized male rat. Endocrinology 117:97-105 10. Laburthe M, Amiranoff B, Boige N, Rouyer-Fessard C, Tetemoto K, Moroder L 1983 Interaction of GRF with VIP receptors and stimulation of adenylate cyclase in rat and human intestinal epi-

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GHRP-6

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thelial membranes. FEBS Lett 159:89-92 Harvey S 1990 Thyrotrophin-releasing hormone: a growth hormone-releasing factor. J Endocrinol 125:345-358 12. Marchant TA, Chang JP, Nahorniak CS, Peter RE 1989 Evidence that gonadotrophin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish. Endocrinology 124:25092518 13. Jones TH, Brown BL, Dobson PRM 1990 Paracrine control of anterior pituitary hormone secretion. J Endocrinol 127:5-13 14. Borges JLC, Uskavith DR, Kaiser DL, Cronin MJ, Evans WS, Thorner MO 1983 Human pancreatic growth hormone-releasing factor-40 (hpGHR-40) allows stimulation of GH release by TRH. Endocrinology 113:1519-1521 15. Leung FC, Taylor JE, Ball CA 1985 Potent interaction between thyrotrophin-releasing hormone (TRH) and human pancreatic growth hormone releasing factor (hpGRF) in stimulating chicken growth hormone (cGH). Domest Anim Endocrinol 2:183-190 16. Rubin AL, Levin SR, Bernstein RG, Tyrell JB, Noacco C, Forsham PH 1973 Stimulation of growth hormone by luteinizing hormone releasing hormone in active acromegaly. J Clin Endocrinol Metab 37:160-166 17. Cantalamessa L, Reschini E, Catania A, Guistina G 1976 Pituitary hormone responses to hypothalamic releasing hormone in acromegaly. Acta-Endocrinol-(Copenh) 83:673-679 18. Codd EE, Aloyo VJ, Walker RF 1990 A non-opioid pattern characterizes inhibition of growth hormone releasing peptide binding by dynorphin-related peptides. Neuropeptides 15:133-137 19. Bercu BB. Weideman CA. Walker RF 1991 Sex differences in growth hormone secretion by rats administered growth hormonereleasing hexapeptide. Endocrinology 129:2592-2598 20. Wehrenbere WB. Brazeau P. Luben R. Line N. Guillemin R 1983 A noninva&e functional lesion of the hypoyhalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology 36:489-491 11.

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21. Wehrenherg WB, Bloch B, Ling N 1985 Pituitary secretion of growth hormone in response to opioid peptides and opiates is mediated through growth hormone-releasing factor. Neuroendocrinology 41:13-X 22. Robbrecht P, Coy DH, Waelbroeck M, Heiman ML, DeNeef P, Camus J-C, Chrostophe J 1985 Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr’, D-Arp2)GRF (l-29)-NH2 as a GRF antagonist on membranes. Endocrinology 117:1759-1764 23. Boockfor FR, Hoeffler JP, Frawley LS 1985 Cultures of GH, cells are functionally heterogeneous: thyrotropin-releasing hormone, estradiol, and cortisol cause reciprocal shifts in the proportions of growth hormone and prolactin secretors. Endocrinology 117:418420 24. Humphries J, Wan Y-P, Folkers K, Bowers CY 1978 Inhibitory analogues of luteinizing hormone-releasing hormone having D aromatic residues in positions 2 and 6 and variations in position 3. J Med Chem 21:120-126 25. Jansson J-O, Downs TR, Beamer WG, Frohman LA 1986 The dwarf “little” (LIT/LIT) mouse is resistant to growth hormone releasing peptide (GHRP-6) as well as to GH-releasing hormone (GRH). Endocrinologv (~~~~1)118:397 26. Edwards CA, DiegueiC, Scanlon MF 1989 Effects of hypothyroidism, tri-iodothyronine, and glucocorticoids on growth hormone responses to growth hormone-releasing hormone and His-D-TrpAla-Trp-D-Phe-Lys-NH*. J Endocrinol121:31-36 27. Katakami H, Downs TR, Frohman LA 1986 Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism. J Clin Invest 77:1704-1711 28. Zeytin GN, Gick GG, Brazeau P, Ling N, McLaughlin M, Bancroft C 1984 Growth hormone (GH)-releasing factor does not regulate GH release or GH mRNA levels in GHa cells. Endocrinology 114:2054-2059

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