The galanin antagonist, M-15, inhibits growth hormone release in rats

Peptides,Vol. 14, pp. 633-636, 1993

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The Galanin Antagonist, M- 15, Inhibits Growth Hormone Release in Rats S T E V E N M. G A B R I E L , l A L E X A N D E R R I V K I N A N D J O H N M E R C A D O , JR.

Department of Psychiatry, Mount Sinai School of Medicine. New York, N Y 10024 Received 9 October 1992 GABRIEL, S. M., A. RIVKIN AND J. MERCADO, JR. The galanin antagonist, M-15. inhibits growth hormone release in rats. PEPTIDES 14(3) 633-636, 1993.--The neuropeptide, galanin, has been implicated in the regulation of rat growth hormone (rGH) release. In the present study, adult male rats were implanted dually with cannulae to the lateral cerebral ventricle and the right atrium. After surgical recovery, rats were infused with M- 15, a specific galanin antagonist, into the lateral ventricle. During the course of this brain infusion, rats were subjected to serial blood sampling with red cell and artificialplasma replacement under stress-free conditions. Plasma was saved for rGH assay. Treatment with M- 15 reduced rGH pulse amplitude and pulse frequency when compared to vehicle-infusedcontrols. These data suggest that brain galanin participates in the ongoing stimulation of pulsatile rGH release in the adult male rat. Galanin

M-15

Growthhormone

Pulsatilehormone

GALANIN is one in a series of peptides isolated by Tatemoto and co-workers in the early 1980s from porcine intestine by virtue of its carboxyl terminus amidation (15). As a reflection of its widespread distribution, it has since been implicated in a variety of regulatory functions, including the control of rGH release (4,8,12-15). The intraventricular administration of antisera to galanin inhibits rGH release (12). Such passive immunization studies presumably work by sequestration of released peptide and thus reduce endogenous peptide binding at galanin receptors. The development of specific galanin receptor antagonists allows for a more direct test of the role of galanin receptor stimulation in the central regulation of rGH release (1). In the following study, rats were administered the galanin antagonist, M- 15, and subjected to repetitive blood sampling for analysis of pulsatile rGH release in a manner parallel to previous passive immunization studies (12).

Anterior pituitary gland

Antagonist

one sitting. Surgery was performed under combined ketamine (50 mg/kg, IM, Parke-Davis, Morris Plains, N J) and pentobarbital (25 mg/kg, IP) anesthesia supplemented with inhaled methoxyflurane (Pitman-Moore, Rundelein, IL). Lateral cerebral cannulations were placed with stereotaxic coordinates (-0.5 mm bregma, + 1.6 mm lateral, 4.5 mm below skull surface) that were verified histologically during technician training and accompanying animal euthanasia using materials from Plastics One (Roanoke, VA). The indwelling right atrial catheters (0.025 in. i.d., Silastic, Dow-Corning, Midland, MI) were placed via the jugular approach using previously described methods (6). After surgery, rats were housed in individual cages within monitoring chambers that were light controlled (12:12 h, light:dark, lights on at 0700 h), ventilated (25 + I°C), sound reduced, and adapted with a swivel through which tubing could be inserted to allow remote brain infusions and venous blood sampling in freely moving unstressed animals (6). Robust rats that regained preoperative body weight were studied 10 to 14 days after surgery. Additional protocols could commence a week later if venous catheter patency was maintained. The galanin antagonist, M-15, was synthesized with a Milligen/Biosearch FMoc Synthesizer and purified to greater than 95% purity. Extension tubing was attached to the ventricular (0.12 ~tl/cm i.d., #MF-5164, Bioanalytical Systems, Lafayette, IN) and venous devices (0.020 in. i.d., Tygon #S-54-HL) at 0730

METHOD Rats were maintained within an accredited vivarium and treated within institutional guidelines. Male Sprague-Dawley (CD) rats weighing 225 to 250 g b.wt. were purchased from Charles River (Wilmington, MA). Animals were handled daily, provided chow and water ad lib, and allowed at least 3 days to acclimate to our colony before surgery. Intracerebral and right atrial cannulations were performed under sterile conditions at

1Requests for reprints should be addressed to Steven M. Gabriel, Ph.D., Department of Psychiatry, Bronx V.A. Medical Center, 130 W. Kingsbridge Rd., Bronx, NY 10468.

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FIG. 1. Representative individual rGH pulse profiles for adult male rats intraventricularly infused with CSF or the galanin antagonist, M-15. The left panels show rGH secretory patterns for rats infused with CSF while the right panels show rGH secretory profiles for rats infused with M-15 peptide. The top two panels show the profiles for a single rat subjected to infusion and blood sampling on two separate occasions--M-15 peptide shown on the top right, followed 1 week later by CSF shown on the left. The arrows at the top of each profile denote significant PC-PULSAR detected pulses.

h. A Bioanalytical Systems C M A - 1 0 0 syringe p u m p delivered artificial cerebrospinal fluid (CSF, 150 m M Na, 2.9 m M K, 2.3 m M C a , 2.3 m M M g , 0.5 m M P , 130 m M C I , 21 mA4 HCO3) or CSF-dissolved peptide. The CSF dead space infusion began at 0830 h (0.34 gl/min). At 0930 h, M-15 peptide or CSF was

delivered at a rate o f 0.139 ~zl/min at a concentration of 1.099 ug/#l for 6 h (54.97 ug or 25 nmol, M- 15 mol.wt. = 2199). This dose was based on the limited solubility o f the peptide in CSF, a previous report of the in vivo effects of M-15, a n d preliminary studies that suggested a sixfold increase in the dose for a con-

GALANIN ANTAGONIST INHIBITS rGH

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TABLE 1 EFFECTS OF THE GALANIN ANTAGONIST, M-15, INFUSION ON PULSATILE GH

Treatment

Baseline (ng/ml)

Amplitude (ng/ml)

Pulse Length (min)

Pulse Frequency (No./h)

CSF [9] M-15 [10]

16.59 +_4.04 20.41 +_6.35

112.36 +_ 18.75 54.81 +_ 17.73"

72.85 _+ 8.68 71.04 ___15.38

0.406 _+0.058 0.231 +_0.053*

Number of subjects in brackets. Values represent mean +_-SEM of PC-PULSAR derived parameters. * Denotes p < 0.05 vs. CSF.

tinuous infusion (1). Blood sampling began at 1000 h at a rate of 0.5 ml blood every 10 min for 6 h using a Gilson Minipulse3 Peristaltic pump (1 ml/min). Plasma was separated from the red cells, placed into vials containing 12.5/A 10% EDTA and 12.5 ~tl aprotinin (Sigma, St. Louis, MO), vortexed, and stored at - 8 5 ° C for later assay. Red cells were resuspended in artificial plasma (50 U/ml heparin, 5% human serum albumin in saline) and returned on the next sample. Plasma rGH concentrations were determined in duplicate 25-#1 samples using the materials provided by the National Hormone and Pituitary Program (anti-rGH-s-4 and rGH-RP-2; sensitivity = 2 ng/ml; EDs0 = 25 ng/ml; inter- and intra-assay coefficients of variations = 10% and 11%, respectively). Peak values of greater than 100 ng/ml were reassayed at 5 gl volume. Pulsatile hormone data were analyzed using PC-PULSAR version 3.0 (Gitzen and Ramirez, Urbana, IL) with peak determination parameters described previously (6). Treatment effects on individual pulse parameters were analyzed using analysis of variance for unequal sample sizes (p < 0.05 significance level). RESULTS

Figure 1 shows representative rGH pulse profiles for rats infused with either CSF or M-I 5 peptide. The left-hand panels show rGH secretory profiles for rats infused with CSF, while the right-hand panels show the rGH secretory profiles for rats infused with the M- 15 peptide. The CSF-infused rats displayed the high amplitude rGH secretory pulses with low interpulse baseline rGH secretion typical of adult male rats (3). The infusion of the galanin antagonist reduced these pulses and their frequency. Table 1 summarizes the effects of CSF or M-15 infusion on PC-PULSAR-derived rGH pulse parameters. This data summarizing 19 profiles represents data from 11 rats, six of which were subjected to repetitive sampling two or three times with at least 1 week recovery between protocols. Pulses of rGH in rats infused with CSF were approximately 100 ng in amplitude, occurred approximately every 2.5 to 3 h, and were interspersed with low or undetectable baseline or interpulse rGH release. In contrast, rats infused with M-15 peptide had pulse amplitudes and pulse frequencies that were significantly reduced approximately 50% compared to controls. Baseline rGH release and pulse length were not different between CSF- and M- 15 peptideinfused rats. DISCUSSION

The blockade of neuronal activity or brain output measures with specific receptor antagonists is a hallmark for demonstrating physiological significance of a biological signal candidate. This criteria has been difficult to achieve for many peptides, because

of the lack of specific antagonists to protein-derived molecules. Consequently, the sequestration of endogenously released peptide with specific peptide antisera represents a useful approximation. Previous studies have demonstrated that centrally administered galanin antisera inhibit the pulsatile release of rGH (12). The present data find similar effects with a centrally infused antagonist to the galanin receptor, M-15. Galanin is synthesized and released from both hypothalamic and pituitary tissues (5,9,11). Accordingly, stimulation by galanin o f r G H release, or its interaction with GH-releasing factor (GRF) to stimulate rGH release, have been reported to be localized to both neuroendocrine sites (4,14). To date, however, galanin receptors have not been detected in the anterior pituitary gland (7,8). Because of the potential for damage to hypothalamic sites from intracerebral cannulation and the multiple sites within the hypothalamus that regulate rGH release, the present study infused the galanin antagonist to the ventricular space. This method of administration has been effective in other neuroendocrine studies (12-14). Therefore, the inhibition of pulsatile rGH release by M-15 in the present study cannot be ascribed with certainty to a direct action within the hypothalamus. In addition to its intrinsic localization within the hypothalamus, galanin localizes within noradrenergic and cholinergic projection neurons that are believed to regulate rGH release (3,15). It also cannot be precluded that the antagonist did not reach the anterior pituitary in sufficient concentrations to affect the somatotroph. Regardless of its site of action, the effect of M- 15 to inhibit pulsatile rGH release likely is due to its specific blockade of galanin receptors (1,2). Thus, it seems probable that galanin actively participates in the ongoing initiation of rGH pulsations. The decline in rGH pulse amplitude during M-15 infusion suggests that galanin may act centrally to stimulate G R F release or interact at the anterior pituitary to potentiate the effect of GRF. Because the antagonist also diminished pulse frequency, it is possible that galanin acts at a central site to stimulate rGH pulse generation, possibly through G R F stimulation or somatostatin inhibition (3). Since the antagonist infusion did not completely abolish rGH secretion, it is also possible that not all galanin receptors relevant to rGH secretion were blocked. The peptide, M- 15 or galanitide, is a hybrid of galanin( 1-13) coupled to substance P(5 - 11). It has an ICso for displacing ventral hippocampal ~2Sl-galanin binding of approximately 0,1 nM, antagonizes galanin's effect on hippocampal acetylcholine release, and blocks galanin-facilitated spinal reflexes (1). Two other chimeral peptides, M-40, which is galanin(1-12) coupled to the idealized a-helix, Pro-Pro-Pro-[Ala-Leu]z-Ala-NH2, and C7, which is galanin( 1-13) coupled to spantide, are less characterized but appear to be more potent displacers at the galanin receptor (2). The c~-helical nature of C-terminal fragment appears to allow

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GABRIEL, R I V K I N A N D M E R C A D O

binding of the biologically active N-terminal galanin fragment without activating the receptor (2). The fact that all these peptides block galanin receptors and receptor-mediated effects despite differences in the carboxyl terminus chimer suggests that their actions are due to galanin receptor antagonism and not due to a specific effect of the carboxyl terminus chimer at another receptor (10).

ACKNOWLEDGEMENTS The authors would like to thank Dr. Tamas Bartfai and colleagues for their most generous gift of the galanin antagonist, M-15, that was used for preliminary observations, and Dr. Salvatore Raiti for his donation of the growth hormone assay reagents. We would also like to acknowledge the excellent technical assistance of Ms. Debra Pearlman. This work was supported by grant MH-45480 (S.M.G.).

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