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Division of Neurophysiology, National Institute for Medical Research, London, NW7 1AA, United Kingdom
Address all correspondence and requests for reprints to: Dr. Gregory B. Thomas, Division of Neurophysiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom. E-mail: g-thomas{at}nimr.mrc.ac.uk
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Introduction |
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Initial studies with the synthetic hexapeptide GHRP-6 suggested that this compound acted primarily on the pituitary gland and was absolutely specific for GH release (6). More recent studies have qualified both of these assumptions. In conscious animals, the GH responses to GHRPs are complex and can best be explained by effects exerted at both pituitary and hypothalamic sites, interacting with both GRF and somatostatin (1, 7, 8, 9). Furthermore, when more recently developed GHRP analogs were tested in man, small but consistent elevations in ACTH and cortisol were observed (3, 10, 11). The clinical significance of these small rises in cortisol is unclear, although they are consistently seen with a variety of GHRP analogs and in different species (12, 13, 14, 15).
GHRPs do not release glucocorticoids from the adrenal glands directly, but via stimulation of ACTH secretion (16), and several lines of evidence suggest that this effect is exerted indirectly via a hypothalamic action. For example, GHRPs do not release ACTH directly from isolated pituitaries (17, 18), nor do they synergize with GRF to release more ACTH in vivo as they do to release GH (19). Furthermore, the ACTH response to GHRPs in vivo is abolished if the pituitary stalk is transected (15). As one in vivo effect of GHRP on GH release is to release endogenous GRF (20), which it then synergizes with at the level of the pituitary to release GH (7, 8), we reasoned that GHRPs might have an analogous action on the hypothalamo-pituitary-adrenal (HPA) axis, by stimulating the release of, or by synergizing with, one or other of the endogenous ACTH secretagogues.
ACTH release is primarily controlled by CRH and arginine vasopressin (AVP) (21). Both peptides stimulate ACTH release when given alone and produce a synergistic response when given together (22). We have now explored the effects of giving GHRP-6 alone or in combination with CRH or AVP on ACTH, corticosterone, and GH release in chronically catheterized conscious rats. Some of these results has been presented in abstract form (23).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal experiments
Exp 1. The aim of this experiment was to ascertain whether
administration of GHRP-6 increased plasma corticosterone concentrations
in the rat and to compare the response to that of CRH. Two group of
four rats that had been catheterized 6 days previously were randomly
injected iv at 1000 h with either 10 µg GHRP-6 or saline (100
µl), and blood samples (20 µl) were collected 15 and 0 min before
and 15, 30, 45, and 60 min after injection. The same rats were injected
again at 1500 h, but with the treatments reversed. On the
following day, using the same injection and sampling protocol, the rats
were randomly injected with either 2 µg CRH or saline. At the end of
the experiment, the diluted plasma aliquots were assayed for
corticosterone by RIA.
Exp 2. The aim of this experiment was to characterize the ACTH response that underlies the increase in plasma corticosterone concentrations induced by GHRP-6 in the rat. A group of 12 rats was implanted with jugular venous catheters under halothane anesthesia. After 2 days of recovery, all animals were connected to the computer-controlled blood-sampling apparatus and injected iv with 10 µg GHRP-6. Beginning 1 h before injection, serial blood samples (30 µl) were withdrawn every 10 min for 3 h and collected into ice-cold tubes containing a 70-µl solution of heparinized saline (5 U/ml), 50 mM EDTA, and 0.1 trypsin inhibitor unit aprotinin (Sigma Chemical Co., St. Louis, MO). The samples were centrifuged at 4 C, and the plasma supernatants were divided into aliquots and stored at -20 C until assayed for ACTH and GH. Samples collected at -10, 0, 20, 30, 40, and 50 min relative to the time of injection were also assayed for corticosterone.
Exp 3. The aim of this experiment was to determine whether GHRP-6 interacted with CRH or AVP to release ACTH or GH in the rat. As the results from Exp 2, begun 2 days after surgery, showed that some rats still had elevated plasma ACTH and corticosterone concentrations before GHRP-6 administration, all rats were left for 4 days to acclimatize to the automatic blood-sampling system before the beginning of this experiment. Groups of rats (n = 5–11) were then randomly injected iv with 10 µg GHRP-6, 2 µg CRH, 0.5 µg AVP, or saline (100 µl), alone or in combination. Blood samples were collected as before, and the plasma supernatants were assayed for ACTH and GH.
Peptides
GHRP-6 was provided by Ferring (Malmo, Sweden). Rat CRH and AVP
were purchased from Bachem (Saffron Walden, UK). All peptides were
dissolved in sterile heparinized (5 IU/ml) saline immediately before
injection.
RIAs
The concentration of ACTH was measured in plasma by direct RIA
(26), using an antiserum (no. 8514/2) supplied by Dr. G. B. Makara,
Institute of Experimental Medicine (Budapest, Hungary). Rat ACTH
(Peninsula Laboratories, St. Helens, UK) was used as the assay
standard. Under our sampling conditions, the sensitivity of the assay
was 40 pg/ml. Rat GH concentrations were measured in diluted plasma
samples by RIA as previously described (24), using reagents generously
supplied by the NIDDK (Bethesda, MD). The sensitivity of the assay was
0.2 ng/ml, and results are expressed in terms of the reference
preparation rGH RP-2. Plasma corticosterone concentrations were
measured using a double antibody RIA kit purchased from ICN Biomedicals
(High Wycombe, UK).
Statistical analysis
All data are shown as the mean ± SEM, and
statistical comparisons were made using ANOVA followed by Duncan’s
multiple range test when a significant (P < 0.05)
interaction was found.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). On the following day, the same rats exhibited a
similar sized increase in corticosterone levels after the
administration of 2 µg CRH, whereas saline injections had no
effect.
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), which revealed a highly significant negative
correlation (r = -0.89; P < 0.001).
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, in which animals with high initial plasma corticosterone levels (>700
ng/ml; n = 6) were analyzed separately (Fig. 3b) from those with
low initial plasma corticosterone levels (<350 ng/ml; n = 6)
before GHRP-6 injection (Fig. 3a). Both plasma ACTH and corticosterone
levels increased markedly (P < 0.001) in response to
GHRP-6 in those rats that had low corticosterone levels 1 h before
GHRP-6 administration (Fig. 3a), whereas in the rats with high initial
corticosterone levels (Fig. 3b), plasma ACTH and corticosterone levels
showed a significant decrease (P < 0.05) 20–30 min
after GHRP-6 injection. GHRP-6 induced GH release in all rats, but the
animals with low initial corticosterone levels exhibited a much greater
(P < 0.01) GH response than those with high initial
corticosterone levels (
GH, 119 ± 27 vs. 29 ±
7 ng/ml; bottom panel, Fig. 3, a vs. b).
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).
The administration of GHRP-6, CRH, or AVP significantly
(P < 0.05) increased plasma concentrations of ACTH.
The mean data are shown in Fig. 4, and the net changes in ACTH release
(ACTH response above baseline) are shown in Fig. 5.
Both GHRP-6 and CRH when given individually released similar amounts of
ACTH, and their combination did not increase plasma ACTH levels beyond
the response to CRH alone (ACTH, 1570 ± 207 vs.
1714 ± 245 pg/ml). However, the combination of GHRP-6 and AVP
produced a significantly (P < 0.05) greater increase
in ACTH than the effects of AVP alone (ACTH, 5587 ± 669
vs. 2338 ± 451 pg/ml). Saline injection had no effect
on plasma ACTH levels.
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and 7. As expected, GHRP-6
stimulated GH release 6- to 7-fold, whereas saline injections had no
effect. Injection of AVP alone had no significant effect on plasma GH
concentrations, whereas CRH alone inhibited (P < 0.05)
GH release for up to 90 min (pre- vs. 40 min post-CRH;
11.9 ± 3.8 vs. 1.7 ± 0.4 ng/ml). When given in
combination with GHRP-6, neither AVP nor CRH affected the GH response
compared with the effects of GHRP-6 alone (Figs. 6 and 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have now documented the increase in plasma ACTH after GHRP-6 treatment in the rat, confirming data from other secretagogues used in other species (11, 12, 14). In one experiment performed only 2 days after surgery, we found that ACTH release in response to GHRP-6 was highly variable and dependent on the prevailing activity of the HPA axis. Rats with high initial corticosterone levels, reflecting an underlying pulsatility of ACTH secretion before GHRP-6 administration, did not show an increase in ACTH release in response to GHRP-6, whereas those rats with lower initial corticosterone and ACTH levels did respond. Much less variability in the ACTH response to GHRP-6 was seen in animals left for a longer recovery period after surgery and in animals that had much lower ACTH levels at the beginning of the experiments. It would, therefore, seem likely that high circulating levels of glucocorticoids feed back to reduce the ACTH response to GHRP-6, as they do for other ACTH secretagogues (27).
Although all rats responded with an increase in GH, this was also blunted in those animals with high initial corticosterone levels. This is in good agreement with other data showing that the GH response to GHRP-6 is significantly reduced after chronic dexamethasone treatment in rats (28) and that the GH response to a low dose (0.2 µg/kg) of L-692,429 is attenuated after glucocorticoid therapy in man (29). Cushing patients also show a reduced response to GHRP-6 (30), but Giustina et al. (31) found that the GH response to hexarelin was not affected in acromegalic patients with glucocorticoid excess. Much less is known about glucocorticoid feedback on GHRP-induced activation of the HPA axis in man, because the rises in cortisol secretion are of much smaller magnitude than those for GH, and their significance is usually discounted. Nevertheless, as GHRP-6 is clearly an ACTH secretagogue in man (3, 10, 11), its actions on this axis are likely to be subject to physiological feedback regulation by glucocorticoids.
The mechanism of glucocorticoid feedback could be exerted at both the pituitary and hypothalamic levels. It has been suggested that elevated glucocorticoids may reduce GH release in rats by stimulating somatostatin release from the hypothalamus (32, 33). Furthermore, central somatostatin has been shown to inhibit the effects of GHRP-6 on GH release (34), whereas antibodies to somatostatin increase the GH response to GHRP-6 (7). Thus, the reduced GH response to GHRP-6 in rats with high circulating corticosterone levels may reflect an increased somatostatin tone in these animals.
As GHRPs do not release ACTH directly, their most likely mechanism of action is to stimulate the release of and/or synergize with endogenous ACTH secretagogues, such as CRH or AVP. We, therefore, tested the effects of GHRP-6, given alone or in combination with these peptides, on both ACTH and GH release. We found a markedly greater increase in plasma ACTH when GHRP-6 was given in combination with AVP, but not when it was given in combination with CRH. This interaction was specific for ACTH, because in the same experiments we found no synergism on GH release when GHRP-6 was given together with AVP or CRH. Although our results identify a synergism between GHRP-6 and AVP on ACTH release, they do not necessarily imply a direct interaction between these peptides. Although GHRPs are not direct ACTH secretagogues (17, 18), the possibility that GHRP-6 amplifies the responses to AVP or CRH at the level of the corticotroph has not been tested. However, we have not observed any additive effect of GHRP-6 on AVP- or CRH-induced ACTH release from cultured rat pituitary cells in vitro (Samsø-Schmidt, L., and I. C. A. F. Robinson, unpublished results), suggesting that this interaction with AVP must occur at the hypothalamic level.
It is well established that CRH and AVP synergize at the pituitary to release ACTH in the rat (22). The most logical explanation for our data, therefore, is that GHRP-6 stimulates CRH release, which then synergizes with AVP at the pituitary. Hickey et al. (12) also considered CRH release induced by GHRPs to be one of several possible mechanisms for the central effects of nonpeptide GH secretagogues to release ACTH in dogs. Our data, however, do not exclude the possibility that GHRP-6 could also release AVP, but we were unable to demonstrate any synergism between GHRP-6 and CRH in these studies. It is also possible that GHRP-6 may interact with ACTH secretagogues other than AVP or CRH (e.g. oxytocin or catecholamines) or may even act via an unidentified factor, as has been postulated for its GH-releasing activity (7).
There is a strong precedent for our hypothesis that GHRP-6 acts within the hypothalamus to release an ACTH secretagogue such as CRH. It is now well established that an important action of GHRPs to release GH involves a hypothalamic effect to release GRF, which then synergizes with GHRPs at the level of the somatotroph to release GH (7, 8). There is direct evidence for release of GRF into hypophysial portal blood after hexarelin administration (20), and recent preliminary evidence from sampling four sheep using this model has confirmed a rise in CRH levels in portal blood after GHRP-6 administration (Guillaume, V., C. Oliver, G. B. Thomas, and I. C. A. F. Robinson, unpublished results).
A dependence on the endogenous peptides governing normal ACTH secretion could also explain why the ability of GHRPs to release ACTH is most manifest when the underlying activity of the HPA axis is low. A rise in glucocorticoid tone would then blunt the effect of GHRP-6 by negative feedback effects on CRH or AVP (27). In the rats with high ACTH levels before injection, administration of GHRP-6 was associated with a transient fall in ACTH. This could reflect an inhibitory effect of GHRP-6 under conditions of high CRH tone. However, an alternative explanation could simply be that the fall in ACTH levels reflects the negative feedback action of the high circulating corticosterone levels, which masks the initial stimulation by GHRP-6.
ACTH-releasing activity has been demonstrated for all classes of peptide or nonpeptide GHRPs reported to date, and these activities are probably mediated via the recently described GHRP receptor (4, 5). Although this receptor has been localized to the hypothalamus in man and swine (5), its distribution in the rat hypothalamus is not yet known. Our data suggest that it might be profitable to look for GHRP receptor expression in the parvocellular region of the paraventricular nucleus (PVN), which is the site of the cell bodies expressing CRH and AVP (35). On the other hand, the effect of GHRPs need not be exerted directly on CRH cell bodies. GHRPs activate Fos protein expression and electrical activity in neurons in a restricted area of the arcuate nucleus, and no GHRP-6-induced Fos protein expression was seen in the PVN (36). However, many of the arcuate cells activated by GHRP-6 are neuropeptide Y (NPY)-positive cells (37), and it is known that the PVN receives a major projection from arcuate NPY neurons (38). It is, therefore, conceivable that a single action of GHRPs on NPY cells in the arcuate nucleus could affect both arcuate GRF neurons and PVN CRH neurons.
Although combinations of CRH or AVP with GHRP-6 had no effect on the ability of the latter to release GH, we noted that injections of CRH alone produced a marked suppression of basal GH release. Central administration of CRH results in an inhibition of spontaneous GH secretion in the rat (39, 40), although in both of these studies, peripheral injections of CRH did not lower mean GH levels. The only obvious difference between our study and those previously reported is that our basal GH levels were very much lower [e.g. 12 vs. 50 ng/ml (39) or 190 ng/ml (40)], which may have made the inhibition of basal GH release easier to detect. It has also been shown that CRH treatment blunts the nocturnal rise in GH secretion in man (41). These inhibitory effects of CRH may well be mediated by a direct stimulation of somatostatin release (40, 42, 43).
These studies have largely focussed on the mechanism of acute activation of the HPA axis by GHRP-6 in the rat. It is clear that the GH-releasing effects of GHRPs are highly dependent on the pattern of exposure to secretagogues and can fade markedly with continuous exposure (44, 45). As glucocorticoids themselves can inhibit the growth-promoting activity of GH (33), it would be desirable, at least on theoretical grounds, to attempt to minimize the ACTH-releasing activity of GHRPs. On the other hand, the changes in adrenal steroid levels are relatively small, and it is unclear whether they persist with continued GHRP-6 treatment or will also desensitize in parallel with GH, as has been shown for nonpeptide GHRPs in man (45). Whatever the biological significance of GHRP-induced activation of the HPA axis, it is important to understand the hypothalamic mechanisms involved, as they will presumably be shared by the unknown endogenous ligand(s) for the recently cloned GHRP receptor (5).
In conclusion, we have shown that GHRP-6 mediates the release of ACTH in conscious rats, and that the magnitude of the release depends on the prevailing activity of the HPA axis. GHRP-6 increases the effects of AVP on ACTH release, and we suggest that this may be a hypothalamic effect via the stimulation of CRH, which then synergizes with AVP at the corticotroph to release ACTH. Furthermore, these effects are probably subject to regulation by glucocorticoids.
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Acknowledgments |
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Received November 1, 1996.
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