Cholecystokinin antagonist, proglumide, stimulates growth hormone release in the rat

J. Biosci., Vol. 15, Number 1, March 1990, pp. 17 21. Printed in India. Cholecystokinin antagonist, proglumide, stimulates growth hormone release in the rat E. VIJAYAN* and S. M. McCANN Department of Physiology, UTHSC Southwestern Medical Center, Dallas, Texas 75235, USA *J. C. Bose School of Life Sciences, Pondicherry University, Pondicherry 605 104, India MS received 18 September 1989; revised 9 April 1990 Abstract. Previous studies have revealed a stimulatory action of cholecystokinin on growth hormone release in the rat. To evaluate the physiologic significance of these effects we employed the cholecystokinin antagonist, proglumide and injected it intravenously and intraventricularly (third cerebral ventricle, 3V) to determine its actions on growth hormone. The experiments were performed in conscious, freely moving rats with indwelling cannulae in the 3V and/or external jugular vein. Intraventricular injection of 2 or 10 µg of proglumide significantly elevated plasma growth hormone concentrations in intact and castrated male rats and in ovariectomized females. Intravenous injections of 10 or 100 µg of proglumide were also effective in elevating growth hormone in a dose-related manner. Surprisingly, the response to the lower dose given intraventricularly was somewhat greater than that of the higher dose. We speculate that these stimulatory effects of proglumide given intraventricularly are due to the agonist action of proglumide at these doses since action of cholecystokinin itself is to increase plasma growth hormone following its intraventricular injection. The studies therefore do not establish a physiologically significant growth hormone-releasing action of brain cholecystokinin but provide more evidence that activation of cholecystokinin receptors in the brain can induce a stimulation of growth hormone release either by activation of the release of growth hormone-releasing hormone or by inhibition of the release of somatostatin or by a combination of these two actions. Keywords. Plasma growth hormone; third ventricle cannulae; hypothalamus; cholecystokinin-antagonist; cholecystokinin-receptors. Introduction The release of growth hormone (GH) from the anterior pituitary is regulated mainly by two hypothalamic peptides, GH-release inhibiting hormone (SRIH, somatostatin) (Reichlin, 1983) and GH-releasing hormone (Guillemin et al., 1982; Rivier et al., 1982). Lesion and stimulation studies have revealed that the ventromedial nucleus (VMH) and the arcuate nucleus (ARC) of the hypothalamus are important structures in regulating GH release (Rechlin, 1985) and immunoreactive cholecystokinin (CCK) neuronal cell bodies were demonstrated in the VMH, the ARC and the medial perifornical region of the lateral hypothalamus in the rat (Straus et al., 1978) which suggested a possible functional role for CCK in the hypothalamus (Dockray, 1983). To test this possibility, CCK-8 was injected into the 3V of ovariectomized, conscious rats. It produced significant elevations in plasma GH levels (Vijayan et al., 1979). Since there was no effect on release of GH by To whom all correspondence should be addressed. Abbreviations used: GH, Growth hormone; VMH, ventromedial nucleus; ARC, arculate nucleus; CCK, cholecystokinin; LH, luteinizing hormone. 17

18 Vijayan and McCann pituitaries incubated in vitro, we concluded that this effect was mediated by a hypothalamic action. Proglumide, a derivative of glutaramic acid, is a selective antagonist for CCK receptors (Hahne et al., 1981; Chiodo and Bunny 1983; Hsiao et al., 1984; Watkins et al., 1984). The availability of such specific antagonists provides a powerful tool to further delineate the physiological role of CCK in the control of GH secretion. Consequently, we investigated the effect of intraventricular or intravenous injection of proglumide on the release of GH in unanesthetized rats. Materials and methods Virgin, female and male Sprague-Dawley rats (Sasco Laboratories, St. Louis, Missouri, USA), weighing 200 240 g, were housed under controlled conditions of light (on 0500 1900 h) and temperature (24 ± 1 C) with free access to rat chow and water. One week after arrival the rats were gonadectomized under ether anesthesia. Groups of intact males and rats 3 weeks after gonadectomy were used for the experiments. One week prior to experimentation a 23 gauge stainless steel cannula was implanted in the 3V and one day prior to experiment an indwelling catheter was placed in the external jugular vein as described previously (Vijayan and McCann, 1978). On the day of the experiment an extenstion of polyethylene tubing (PE 50, 12 long) filled with heparin in 0 9% NaCl was attached to the distal end of the intravenous cannula and the animals were left undisturbed in individual cages for 60 120 min. During this time, a preinjection blood sample (0 8 ml) was withdrawn over a period of 60 s just prior to injection of proglumide or the saline diluent. Proglumide (DL-4-benzamido-N, N-dipropyl-glutaramic acid) was microinjected into the 3V in a volume of 2 µl of saline using a 10 µl Hamilton microsyringe as previously described (Vijayan et al., 1979). The ph of the proglumide solution was similar to that of the physiologic saline. Control rats received an equal volume of saline by the appropriate route of injection. In all cases the injection time was around 60 s and all experiments were performed in the morning between 0830 1100 h. Blood samples (0 8 ml) were collected in heparinized syringes from the external jugular vein at varying intervals (see Results) while the animal was freely moving in the cage. The volume of each blood sample was replaced immediately after bleeding by an equivalent volume of saline. Plasma was separated by centrifugation at low speed at 4 C and stored frozen until the day of assay. Radioimmunoassay Plasma GH concentrations were measured by radioimmunoassay using the kits provided by NIDDK and expressed in terms of the RP-1-NIH-GH standard. To eliminate inter assay variation, all samples from each experiment were run in the same assay in duplicate. The quality of assays was controlled according to the criteria proposed by Rodbard et al. (1961). Data are expressed as mean ± SEM and were analyzed by analysis of variance for repeated measures followed by the Student Neuman-Keuls test.

Proglumide stimulates GH release 19 Results GH release in intact and castrated male rats Intraventricular injection of 2 or 10 µg proglumide produced a significant elevation of plasma GH levels in intact male rats (figure 1A). The lower dose of 2 µg produced a sharp increase, reaching the peak at 30 min. The hormone levels decreased by 60 min but rose sharply again to peak values at 120min after injection. Plasma GH levels rose gradually and remained significantly elevated for the 120 min duration of the experiment following the higher 10 µg dose. Systemic intravenous injection of proglumide at this dose (10 µg) produced a delayed rise in plasma GH levels which became significant at 120 min, whereas the 100 µg dose was already effective at 60 min after the injection and values remained elevated at 120 min (figure 1B). Figure 1. Effects of various doses of proglumide on plasma GH in male rats. (A) Third ventricular injection; (B) intravenous injection. Mean values of plasma GH are shown as symbols in A and by the height of the bars in this and subsequent figures. Vertical lines = SEM. Intraventricular proglumide produced a dose-related increase in plasma GH in castrated male rats (figure 2A). The magnitude of the increase in GH levels were similar to those observed in intact males. Intravenous injection of proglumide in castrated males produced similar increases in hormone levels at 60 and 120 min as seen in the intact rats (figure 2B). Ovairectomized rats Intraventricular injection of 2 µg proglumide had no effect on plasma GH at 15 and 30 min but it increased significantly at 60 and 120 min after injection. The 10 µg dose, however, caused a significant increase only at 30 min following injection (figure 3A). Interestingly, the lower dose of 2 µg produced a greater stimulatory effect than the higher dose as was seen in intact male rats.

20 Vijayan and McCann Figure 2. Effect of various doses of proglumide on plasma GH in castrated males. Figure 3. Effect of various doses of proglumide on plasma GH in ovariectomized rats. Systemic injection, on the other hand, produced a dose-related increase in plasma hormone levels. The lower dose of 10 µg was effective at 30 min while the 100 µg dose was effective at 15 min following injection (figure 3B). Discussion The present results indicate that proglumide is a potent GH secretagogue in vivo in the rat. Interestingly the effect of proglumide on GH release appears to be identical to that of CCK. The dose of proglumide used, however, is much higher than the effective doses of CCK which stimulated GH release after third ventricular injections (Vijayan et al., 1979). Since proglumide is a specific CCK antagonist one would expect a reversal of the CCK effects on hormone release. This was true in the case of luteinizing hormone (LH) and prolactin since proglumide had opposite

Proglumide stimulates GH release 21 effects to those of CCK on plasma levels of LH and prolactin (Vijayan and McCann, 1986, 1987). In contrast, both doses of 3V and intravenous proglumide produced significant increases in GH release. This is very puzzling in view of the stimulatory effect of intraventricular CCK on GH release (Vijayan et al., 1979). Intravenous injection of 10 µg dose produced, time-related, progressive increase whereas 100 µg dose caused significantly higher elevation in plasma GH levels. The responsiveness to intraventricular proglumide was nearly equal in intact males, castrated and ovariectomized rats. However, ovariectomized rats appear to be more sensitive to proglumide after intravenous dose as the hormone levels were significantly higher than in those of intact or castrated animals. Non-specific peripheral effects or the absence of ovarian steroids cannot be ruled out in the case of intravenous doses. In the cases of both LH and prolactin, as the dose of proglumide was increased it no longer had opposite effects to those of CCK, which suggests that high doses of proglumide had an agonist action instead of an antagonist effect. We speculate that the ability of proglumide to elevate plasma GH may reflect a CCK agonist action. Further experiments with a more specific antagonist or with CCK-antiserum will be needed to establish this hypothesis. Proglumide may inhibit the release of somatostatin from neurons near the third ventricle. Alternatively, it may stimulate the release of growth hormone releasing hormone, or both action may occur. Since CCK coexists within and excites midbrain dopamine neurons (Chiodo and Bunny, 1983) and there is an involvment of central dopaminergic systems in pituitary hormone release (Vijayan and McCann, 1978), it would be of interest to ascertain if the actions of CCK and proglumide on GH release may be mediated by the dopaminergic system. Peripheral effects cannot be ruled out in the case of intravenous injections of proglumide. Acknowledgement This research was supported by NIH grant DK 10073. References Chiodo, L. A. and Bunny, B. S. (1983) Science, 219, 1449. Dockray, C. J. (1983) in Brain peptides (eds D. T. Krieger, Μ. J. Brownstein and J. Β. Martin) (New York: John Wiley) p. 851. Guillemin, R., Brazeau, P., Bohlen, P., Esch, F. and Ling, N. (1982) Science, 218, 585. Hahne, W. F., Jensen, R. J., Lemp, G. F. and Gardine, J. D. (1981) Proc. Natl. Acad. Sci. USA, 78, 6304. Hsiao, S., Katsuura, G. and Itoh, S. (1984) Life Sci., 34, 2165. Reichlin, S. (1983) in Brain peptides (eds D. T. Krieger and J. B. Martin) (New York: John Wiley) p. 171. Reichlin, S. (1985) in Textbook of endocrinology (eds J. E. Wilson and D. W. Foster) (Philadelphia: W. B. Saunders) p. 492. Rivier, J., Spiess, J., Thorner, M. and Vale, W. (1982) Nature (London), 300, 276. Rodbrad, D., Rayford, P. L., Cooper and Ross, G. T. (1961) J. Clin. Endocrinol., 28, 1412. Straus, Ε., Muller, J. Ε., Chol, Η., Paronetto, F. and Yalow, R. S. (1978) Proc. Natl. Acad. Sci. USA, 75, 486. Vijayan, Ε., Samson, W. Κ. and McCann, S. Μ. (1979) Brain Res., 172, 295. Vijayan, Ε. and McCann, S. Μ. (1978) Neuroendocrinology, 25, 150. Vijayan, Ε. and McCann, S. Μ. (1986) Brain Res. Bull, 16, 533. Vijayan, Ε. and McCann, S. M. (1987) Life Sci., 40, 629. Watkins, L. R., Kinschek, I. B. and Mayer, D. J. (1984) Science, 224, 395.