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GRH-SOMATOSTATIN-GH |
Department of Biomedical Sciences and Biotechnology (G.T., D.S., M.B., D.C.), Internal Medicine (A.G.), University of Brescia, 25125 Brescia, Italy; and Biomeasure Inc., (M.D.C., J.E.T.) Milford, Massachusetts
Address all correspondence and requests for reprints to: Dr. Andrea Giustina, Endocrine Section, 2a Medicina, Spedali Civili di Brescia, 25125 Brescia, Italy. E-mail: . giustina{at}master.cci.unibs.it
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-cells, with resultant increases in plasma glucose and then insulin. |
Introduction |
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Recently, SS antagonist peptides have been synthesized that exhibit high affinity and receptor subtype selectivity (2, 3, 4, 5, 6). sst2-selective antagonists could be clinically useful in states of decreased GH secretion caused by altered hypothalamic SS activity, such as in patients undergoing chronic corticosteroid therapy. Reportedly, glucocorticoids are potent inhibitors of linear growth in both man and experimental animals when secreted or administered in supraphysiological amounts (7, 8). Long-term high-dose glucocorticoids alter spontaneous GH secretion and blunt the GH response to a variety of physiological and pharmacological stimuli in humans and in rodents (9, 10, 11, 12, 13, 14). The inhibitory effects on GH secretion can be mainly attributed to a glucocorticoid-mediated enhancement of hypothalamic SS release (11, 12, 13), as suggested by the increased hypothalamic SS mRNA found after pharmacological doses of dexamethasone (DEX) (15, 16). Accordingly, the inhibitory effects of glucocorticoid treatment on somatic growth in juvenile male rats is partially reversed by concomitant treatment with SS antibodies (17).
In this paper, we report on two peptides with in vitro properties consistent with pure sst2-selective antagonists, based on their binding profile and ability to counteract SS-induced Ca2+ mobilization in sst2-expressing cells. Furthermore, we evaluated their GH-releasing activity, when administered by different routes, in anesthetized adult rats and in freely moving 10-d-old rats. Both animal models are useful for in vivo studies because, unlike adult conscious rats, plasma GH levels are relatively stable but remain measurable and sensitive to SS-neutralizing agents. In the same experiments, because SS is also an important regulatory peptide for the activity of the endocrine pancreas, we examined the influence on glucose homeostasis and insulin/glucagon secretion. Finally, we describe the effect of these SS-antagonists on the GH response to GHRH after prolonged high-dose DEX treatment in young male rats.
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Materials and Methods |
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Radioligand binding assays.
Membranes for in vitro receptor binding assays were obtained by homogenizing (Polytron setting 6, 15 sec) the CHO-K1 cells, expressing the h-sst subtypes, in ice-cold 50 mM Tris-HCl, and centrifuging twice at 39,000 x g (10 min), with an intermediate resuspension in fresh buffer. The final pellets were resuspended in 10 mM Tris-HCl for assay. For the h-sst1, 3, 4, and 5 assays, aliquots of the membrane preparations were incubated [90 min/25 C with 0.05 nM [125I-Tyr11]somatostatin (SS)-14 in 50 mM HEPES (pH 7.4) containing BSA (10 mg/ml), MgCl2 (5 mM), Trasylol (200 kIU/ml), bacitracin (0.02 mg/ml), and phenylmethylsulfonylfluoride (0.02 mg/ml)]. The final assay vol was 0.3 ml. For the h-sst2 assay, [125I]MK-678 (0.05 nM) was employed as the radioligand, and the incubation time was 90 min/25 C. The incubations were terminated by rapid filtration through GF/C filters (presoaked in 0.3% polyethylenimine) using a Brandel filtration manifold. Each tube and filter were then washed three times with 5-ml aliquots of ice-cold buffer. Specific binding was defined as the total radioligand bound minus that bound in the presence of 1,000 nM SS-14 (for h-sst1–3 to -5) or 1,000 nM MK-678 (for h-sst2).
Calcium mobilization assay.
CHO-K1 cells, expressing the human sst2 or sst5 receptors, were harvested by incubating in a 0.3% EDTA/PBS solution (25 C) and washing twice by centrifugation. The washed cells were resuspended in HBSS for loading of the fluorescent Ca2+ indicator Fura-2AM. Cell suspensions of approximately 106 cells/ml were incubated with 2 µM Fura-2AM for 30 min at 25 C. Unloaded Fura-2AM was removed by centrifugation twice in HBBS, and the final suspensions were transferred to a spectrofluorometer (Hitachi F-2000) equipped with a magnetic stirring mechanism and a temperature-regulated cuvette holder. After equilibration to 37 C, the SS peptides were added for measurement of intracellular Ca2+ mobilization. The excitation and emission wavelengths were 340 and 510 nm, respectively. Validation experiments demonstrated that the biological activity of the various analogs was in keeping with their receptor binding profile.
Rat pituitary primary cell cultures.
Adult male rat pituitaries were washed in PBS, then minced and enzymatically dissociated using 0.35% collagenase (type IA), 0.1% trypsin (both from Sigma, St. Louis, MO) for 10 min at 37 C. Cell suspension was centrifuged and pellet resuspended in low glucose DMEM supplemented with 10% FCS, 2 mM glutamine, and antibiotics. Cells were seeded in 24-well culture plates (Costar, Cambridge, MA), at a density of 100,000 cells/well, in 1 ml culture medium and incubated for 96 h in a humidified atmosphere of 95% air-5% CO2, at 37 C.
Incubations with SS-14 or coincubation experiments with SS-14 and SS antagonists were carried out through 6 h in medium without serum, on groups of four wells for each experimental point; then, growth medium was collected and stored frozen for later GH measurements.
In vivo studies
Animals.
This study was performed according to the Italian ethics legislation governing these experiments (Ministry of Health, Decreto Legge 116/92). Young male Sprague Dawley rats (300–350 g) and 10-d-old neonatal rats were used in this study. Animals were obtained from Harlan Sprague Dawley, Inc. Italy (S. Pietro al Natisone, Italy). Adult rats arrived 2 wk before the experiments. Pups were kept with their mothers and were allowed to remain with dams until 1 h before the experiments. Animals were kept in a temperature-controlled environment (21-23 C) with a 14-h light, 10-h dark cycle and had free access to food and water.
Experiments in anesthetized adult rats.
Rats (350–400 g) were anesthetized with urethane (1.5 g/kg, ip) and fitted with jugular catheters allowing iv treatment as well as blood sampling. SS antagonists were administered 30 min after anesthesia, and blood samples (200 µl) were collected from 0 min up to 45 min after administration. Using this system, we carried out time-course experiments, determined the dose-response curves, and compared the iv, ip, and sc routes.
Experiments in 10-d-old freely moving neonatal rats.
SS antagonist BIM-23627 was tested sc or ip in freely moving 10-d-old rats. Trunk blood was collected from groups of six rats killed by rapid decapitation, 0 min, 10 min, 20 min, and 30 min after drug administration.
Experiments in glucocorticoid-treated rats.
Male adult rats (300–350 g) were treated daily (ip, 0900 h) for 5 d, including the day of the experiment, with either saline or the synthetic glucocorticoid DEX sodium phosphate (40 µg/d). This dose and the duration of the treatment were selected based on previous work of Wehrenberg and colleagues (10). On the day of the experiment, animals were anesthetized with ketamine//xylazine (60 mg/kg//6 mg/kg) and fitted with jugular catheters. Twenty minutes later, after having one control blood sample drawn, six animals of each group were treated with SS antagonist (BIM-23627; 0.2 mg/kg, iv) and six with vehicle only. A maximally effective dose of GHRH 1–29 (1 µg/kg, iv) was administered to all the animals, and subsequent blood samples (200 µl) were drawn at 5 min, 10 min, 15 min, and 30 min after GHRH administration.
Hormones and glucose determination.
After blood samples were collected into EDTA-containing tubes, plasma was separated by centrifugation and stored at -20 C until hormone determination. GH concentrations in plasma and cell culture media were determined, by RIA, with reagents kindly provided by NIDDK (Bethesda, MD; minimum detectable dose, 0.4 ng/ml). Plasma insulin and glucagon levels were measured by commercial RIA kits (ICN Biomedicals, Inc., Asse-Relegem, Belgium), and blood glucose levels were determined using Glucotrend (Roche Diagnostics, Barcelona, Spain).
Drugs
Two sst2 antagonist peptides were selected for the present study: BIM-23627 (MW 1164) and BIM-23454 (MW 1146) (Biomeasure Inc.). Peptides were dissolved in 0.1 M acetate buffer, 0.1% BSA, pH 5.2.
Urethane was purchased from Fluka Chemical Co. (Buchs, Switzerland), ketamine (Inoketam) from Virbac (Milan, Italy), and xylazine (Rompum) from Bayer Corp. (Leverkusen, Germany). DEX sodium phosphate (Decadron) was purchased from Merck \|[amp ]\| Co., Inc. (West Point, PA), and GHRH 1–29 (Geref) was obtained from Serono Laboratories, Inc. (Milano, Italy).
Statistics
Unless otherwise stated, data are reported as mean ± SE. The results were analyzed by one-way ANOVA or multifactorial ANOVA for repeated measures (time course experiments in DEX-treated rats), with statistical significance defined as P < 0.05.
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Results |
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).
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). Conversely, the sst5-preferring agonists, BIM-23206 and BIM-23268, were potent stimulators of Ca2+ mobilization in sst5-expressing cells, but the sst2-preferring analog, BIM-23197, weakly stimulated Ca2+ mobilization in keeping with its affinity for the sst5 subtype (Fig. 1B). The sst2 antagonists, BIM-23454 and BIM-23627, were inactive in stimulating Ca2+ mobilization with the sst2-expressing cells and displayed only slight agonist activity with the sst5-expressing cells in the range of 300-1000 nM. Both BIM-23454 and BIM-23627 inhibited SS-induced Ca2+ mobilization in sst2-expressing cells in a dose-dependent manner, with complete suppression of SS action observed with a 10:1 ratio of either BIM-23454 or BIM-23627:SS (Fig. 2).
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).
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). The minimal effective dose, under these conditions, was 1 mg/kg for BIM-23454 (A) and 0.02 mg/kg for BIM-23627 (B). At the higher dose tested (0.2 mg/kg for BIM-23627 and 2.5 mg/kg for BIM-23454), both peptides increased GH levels about 10-fold vs. basal values within 5 min after administration. The GH-releasing effect of BIM-23454 was relatively brief, and it disappeared within 15 min, whereas BIM-23627 induced a more stable GH increase, lasting through 15 min. Injection of BIM-23627, ip or sc, still significantly increased plasma GH, at the same doses tested iv, although the GH pulses showed lower amplitude (P < 0.05), as compared with the iv route (2-fold vs. basal values, 20 h after injection); and plasma GH levels returned to baseline within 30 h after treatment (Fig. 5, A and B).
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).
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). In saline-treated rats, the peak and subsequent waning GH response to GHRH was increased, but not significantly so, by simultaneous administration of the SS antagonist, BIM-23627. In DEX-treated rats, however, GHRH and BIM-23627, administered together, resulted in a significantly higher GH pulse amplitude and response duration, as compared with GHRH alone. With simultaneous antagonist administration, the peak GH response to GHRH in the DEX-treated group was not significantly different from the peak response of the saline-treated group to GHRH alone. Total GH secretion [area under the curve (AUC)] was also clearly increased by antagonist treatment (Fig. 7B), and significantly so in the DEX-treated groups (P < 0.05). However, because of the higher variability in the saline-treated groups, there were no significant differences between the AUC values of the saline and DEX-treated groups.
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). This effect of BIM-23627 was confirmed by results in freely moving pups, in which 20 min after ip or sc administration (0.2 mg/kg), blood glucose levels were significantly higher, as compared with controls (Fig. 8B).
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).
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). In contrast, BIM-23627 displayed less activity in stimulating the release of insulin (P < 0.1 for 0.2 mg/kg sc or 2 mg/kg ip), and only the maximal dose tested sc (2 mg/kg) caused a significant increase (P < 0.05) vs. saline-treated controls, within 20 min after administration (Fig. 10B).
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Discussion |
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The new peptides BIM-23627 and BIM-23454 have proven to be SS antagonists that selectively bind with high affinity to h-sst2 but are completely inactive in stimulating intracellular calcium mobilization; thus, they act as pure antagonists. These findings distinguish these molecules from many potent analogs that have partial agonist activity both in vitro and, at higher doses, in vivo. In h-sst5-expressing cells, both peptides at high concentrations produced only a weak stimulation of calcium mobilization, in keeping with their low affinity for the subtype-5 receptor. Before proposing clinical evaluation of SS antagonists, a pharmacological characterization of their action in experimental animals is necessary. We here report the effects of BIM-23627 and BIM-23454 on GH release in urethane-anesthetized male adult rats and in freely moving, 10-d-old rats. Both systems are useful for in vivo studies because, unlike adult conscious rats, plasma GH levels are relatively stable but remain measurable. Urethane increases the release of SS from the hypothalamus, thus suppressing pulsatile GH secretion (21). As a consequence, urethane-anesthetized rats have low plasma GH levels and are highly sensitive to SS-neutralizing agents. In contrast, 10 d after birth, the immature pituitary is only partially responsive to endogenous SS, because of the low level of sst2 expression on somatotroph cells (22, 23); however, it has been shown that SS antiserum still effectively increases serum GH concentrations in these rats. We therefore considered this model useful in evaluating the effects of SS antagonist peptides in freely moving animals (24).
The iv administration of both BIM-23627 and BIM-23454 induced reproducible, dose-dependent increases in GH release in urethane-anesthetized rats. The minimal effective dose was significantly higher for BIM-23454 vs. BIM-23627, as expected from the in vitro estimates of the SS antagonist potency in rat pituitary primary cell cultures. The sc or ip injection of BIM-23627, at the same doses tested iv, still significantly increased plasma GH, although to a lesser extent, likely because of decreased bioavailability, as compared with the iv route.
In 10-d-old, freely moving rats, sc or ip administration of BIM-23627 induced a short-lived, small rise in GH levels, despite increasing the dose to 2 mg/kg. The results were similar to those obtained in urethane-anesthetized rats, with no differences in either GH pulse amplitude or kinetics; thus, it is unlikely that urethane anesthesia altered either the uptake or the bioavailability of the drugs.
The inhibitory effect of the short-term administration of glucocorticoids on the GHRH-induced GH release was documented by the lower GH peak concentration in DEX-treated animals, as compared with saline-treated controls, whereas the overall GH response was not significantly affected because of the high variance. The ketamine/xylazine anesthesia used in this study reportedly does not alter pulsatile GH secretion and hypothalamic SS tone (25, 26). In rats treated with saline, the GH response to both GHRH and GHRH + BIM-23627 was highly variable, probably reflecting differences in spontaneous SS tone. Similarly, the enhanced secretion of SS may explain the much lower degree of variability observed in the GH responses of the DEX-treated group. The iv administration of BIM-23627 did not significantly influence the GHRH effect in the saline-treated control animals but significantly enhanced the GH response to GHRH in rats treated with high doses of DEX. These data demonstrate that BIM-23627, a subtype-2 specific antagonist, is able to counteract the increase in hypothalamic SS tone after short-term, high-dose glucocorticoid treatment. The effect of BIM-23627 is slightly lower, as compared with that of SS antiserum, in the same model of DEX-induced suppression of GH-axis (10). However, in the previous study, rats were pretreated with SS antiserum, 30 min before GHRH injection; whereas, in the present study, BIM-23627 and GHRH were given simultaneously. This difference, and the lower stability of small peptides vs. Igs, could partially explain the different results.
In both anesthetized rats and freely moving pups, SS antagonist administration caused modest (but reproducible) increases in blood glucose levels. This effect is unlikely to be related to the increase in GH because, under the same experimental conditions, the GH secretagogue hexarelin did not modify glucose levels, despite potently increasing plasma GH with the same time course (data not shown). SS plays an important role in the regulation of insulin and glucagon secretion from the endocrine pancreas, probably acting through different receptor subtypes. Immunohistochemical studies of rat endocrine islets have demonstrated that -cells strongly express sst2, whereas ß-cells show only modest sst2 immunoreactivity (27). In keeping with this observation, it has been demonstrated that activation of sst5 (but not sst2) inhibits insulin secretion from rat islet cells, whereas glucagon secretion is suppressed by sst2 (18, 19). Thus, sst2-selective antagonists may be hypothesized to neutralize the inhibitory effect of endogenous SS on glucagon secretion without altering insulin secretion. We here report direct evidence that, in freely moving 10-d-old rats, BIM-23627 was highly effective in stimulating glucagon secretion, with consequent increase in plasma glucose and insulin levels. In both urethane-anesthetized rats and freely moving pups, BIM-23627 significantly stimulated insulin secretion but only at the maximal dose tested. This effect is not likely to be attributable to the low affinity of BIM-23627 for the sst5 receptor expressed by the ß-cells, because BIM-23627 is an agonist at this receptor subtype and, if sufficiently potent to activate the receptor, would be expected to suppress insulin secretion. The observed increase in insulin levels is more likely the response to glucagon-induced-hyperglycemia. Alternatively, because sst can inhibit gastric emptying and reduce the rate of perfusion of small intestine (28), the effects on insulin may also be hypothesized to be secondary to stimulatory actions of the sst antagonist on nutrient (i.e. glucose) uptake. This hypothesis is less likely, because we observed a similar effect on insulin levels also in fasted rats (data not shown).
In conclusion, the effects of BIM-23454 and BIM-23627 in rats are consistent with the sst2-selective antagonist activity documented by in vitro studies in h-sst-expressing cells. Both peptides were able to counteract the SS-induced inhibition of GH secretion occurring after urethane anesthesia and under conditions of excess glucocorticoids. Moreover, the sc or ip routes have been shown to require larger doses of the antagonists to induce GH pulses that are comparable with those induced by the iv route, because of the decreased bioavailability. Further in vivo studies with SS antagonists will prove invaluable in dissecting the contribution of the five different receptor subtypes in mediating the many inhibitory effects of SS.
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Footnotes |
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Abbreviations: AUC, Area under the curve; CHO, Chinese hamster ovary; DEX, dexamethasone; SS, somatostatin.
Received August 28, 2001.
Accepted for publication December 3, 2001.
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