Biological properties of salmon calcitonin IV.

In this study we characterized the biological activity of the recently identified salmon calcitonin (sCT) IV, in order to evaluate its potential therapeutic value. In the rat bioassay, sCT IV exhibited a 30% higher hypocalcemic activity than sCT I. The capacity of the molecule to inhibit bone resorption was assessed in vitro by the bone resorbing assay and the pit assay. An inhibitory effect, similar to that of sCT I, was observed in both assays. The interaction of sCT IV with the rabbit CT receptor was also studied. The affinity of sCT IV for the receptor was similar to that of sCT I, as was the potency for stimulating cAMP production. The antigenicity of the two molecules was not identical. Thus, this new CT could represent a useful novel therapeutic agent for the treatment of bone disorders.     

Salmon calcitonin treatment by nasal spray in primary hyperparathyroidism.

The hypocalcemic and hypophosphatemic effect of salmon calcitonin (sCT) given by intranasal (i.n.) spray to 12 patients with histological confirmed primary hyperparathyroidism (1 degree HPT) was studied. The concentration of ionized calcium in whole blood (B-Ca++), serum phosphate (S-P), magnesium (S-Mg), plasma sCT (Pl-sCT), and endogenous CT (hCT) was followed during five 24-hour periods with at least three days between. After period I (control day), 100 IU sCT was given intramuscularly (i.m.) in period II. In periods III-V, either 110, 200, or 400 IU of sCT were given intranasally (i.n.) in randomized order. Although B-Ca++ decreased from the baseline value with all four sCT treatments and at 4.5 hour on the control day (p less than 0.05-0.001), the i.n. sCT treatments had no significant hypocalcemic effect, as the change of the area under the B-Ca++ curve (delta AUC B-Ca++) for the three i.n. treatments was not significantly different from the control period (p less than 0.001, ANOVA). Only the i.m. injection of calcitonin had a calcium-lowering effect (p less than 0.001, ANOVA). Three subjects were considered nonresponders with a decrease in B-Ca++ less than 0.06 mmol/L. S-P decreased within three hours after 200 IU sCT i.n. and 100 IU i.m., but the S-Mg levels showed no consistent changes. The area under the curve for the Pl-sCT levels did not correlate with delta AUC B-Ca++ except for i.m. given sCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salmon calcitonin and cGMP production by human kidney: studies in vivo and in vitro

In order to evaluate whether or not the action of salmon calcitonin (sCT) at the kidney level could be mediated through specific receptors for the hormone, we have studied the effects of sCT infusions on urinary excretion of cyclic nucleotides in humans. Parallel in vitro studies have been conducted by evaluating the effects of sCT on cyclic nucleotide levels in primary cultures of cortical and medullary human kidney cells. In vivo experiments showed that sCT induced an increase in cGMP in human urine, which was rapid and short-lasting, being superimposable on the increase of urinary excretion of calcium and magnesium. The increase of inorganic phosphate urinary excretion was delayed and appeared to parallel that of urinary cAMP. On the other hand, our in vitro experiments showed that sCT stimulated the guanylate cyclase-cGMP system of human kidney cortical cells at nanomolar concentrations, while higher concentrations of the hormone were required to activate the adenylate cyclase-cAMP system. In addition, sCT was not able to significantly modify the cellular levels of either nucleotide in human kidney medullary cells. Present data demonstrated a direct effect of sCT on human kidney cortical cGMP production, while the efficacy of sCT on the kidney cortex adenylate cyclase-cAMP system appears to be delayed and/or reduced.     

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.     

Inhibitory effect of salmon calcitonin on bone resorption: Morphological study of the tibial growth plate in rats

Summary Salmon calcitonin (sCT) at doses of 100 and 50 UI given subcutaneously to growing rats produced in vivo evidence of osteoclastic activity inhibition. Histological assessment was carried out by measuring the perichondrial ring of Lacroix height, and a dose-correlated effect was found. These aspects were coupled with an increase in the osteoclast number and suggested that in studies with bone resorption inhibitors, morphological evaluation based on osteoclasts count is not reliable. The changes of the metaphysis suggested also that sCT affects the activity of hypertrophic chondrocytes of the growth plate. Plasma calcium levels did not differ significantly between treated rats and controls; an increased phosphatemia was observed in sCT-treated animals.     

Prevention of ovariectomy osteopenia in rats after vaginal administration of Hyaff 11 microspheres containing salmon calcitonin

The benzyl ester of hyaluronic acid (Hyaff 11) is a highly mucoadhesive polymer and can be processed into microspheres that can effectively deliver incorporated drugs by closely adhering to the mucosal surface and by protecting the drug from enzymatic inactivation. In this study, Hyaff 11 microspheres have been investigated as novel delivery system for the vaginal administration of salmon calcitonin (sCT) to ovariectomized rats. Moreover, this particular animal model has been used to evaluate the biological activity of the polypeptide after vaginal and I.M. administration by measuring the skeletal effect of sCT. A total of 66 female rats were used: 6 rats served as the basal group; 12 were subjected to sham operation and left without pharmacological treatment; 48 underwent bilateral ovariectomy and these were divided at random into four groups of 12 animals. One group was not submitted to pharmacological treatment; another was treated daily with sCT I.M.; in the other two groups, the hormone was incorporated into Hyaff 11 microspheres and was daily administered by vaginal route as dry powder or dispersed in pessaries. Sixty days after surgery, rats were sacrificed and the proximal third of the right tibia was removed and prepared for histomorphometry. Treatment with sCT, independent of the administration route, largely prevented the bone volume loss of the nontreated ovariectomized group. Prevention mainly depended on the maintenance of the trabecular number and on the increase of the trabecular thickness. Independent of the administration procedure, sCT greatly reduced the increase of tartrateresistant acid phosphatase-positive poly- and, particularly, mononucleated osteoclasts found in the nontreated ovariectomized group. The increase of mineral appositon rate of the nontreated ovariectomized group was significantly prevented in the sCT treated groups. The results demonstrate that vaginal administration of sCT, as well as I.M. administration, prevent bone loss due to ovariectomy mainly by inhibiting osteoclastic resorption.     

Biphasic effect of calcitonin on tartrate-resistant acid phosphatase activity in isolated rat osteoclasts

Tartrate-resistant acid phosphatase (TRAP) has been implicated as being involved in osteoclastic bone resorption, and calcitonin (CT) is known to inhibit the resorptive process. This study investigates the kinetics of CT action on TRAP activity in isolated rat osteoclasts using both biochemical and quantitative cytochemical methods. The latter technique has been developed to detect very small changes in intracellular TRAP activity at the single-cell level. The biochemical study showed that 10(-9) M salmon CT (sCT) decreased TRAP activity in medium throughout the experimental period; TRAP activity in the cells was increased during the first 2 h but subsequently declined and was decreased to a significant level at 6 h. TRAP activity in sCT-treated osteoclasts measured by the cytochemical method showed significant increases within the first hour. This response was dose dependent between 10(-16) and 10(-11) M sCT with EC50 at 8 X 10(-14) M. After 1 h, the initial increase in intracellular TRAP activity in CT-treated osteoclasts was followed by a decline to below control levels, reaching statistical significance at 9 h. Treatment with forskolin (10(-5) M) showed a similar trend, suggesting that this response is mediated by cyclic AMP-regulated phosphorylation events. From these results, we conclude that CT has two actions on TRAP in isolated rat osteoclasts: the first to inhibit its release, the second to inhibit its synthesis and/or increase its degradation.     

Factors influencing the enhanced hypocalcemic action of liposome-entrapped calcitonin

Summary The mechanism whereby liposomal entrapment enhances the hypocalcemic effect of calcitonin (CT) was evaluated in the rat. Phosphatidylcholine (PC) and PC plus cholesterol (PCC) preparations of large multilammelar vesicles (MLV) and small unilammelar vesicles (SUV) were used to entrap human calcitonin (hCT). The effect of each of these preparations was assessed by measuring plasma calcium after their parenteral administration. Changes in calcium were compared with plasma concentrations of liposomal hCT (L-hCT) and free hCT (F-CT). The plasma form of hCT was also evaluated by gel filtration chromatography. Most of the liposomal preparations had a greater hypocalcemic effect than unencapsulated hCT. Following I.V. administration, the MLV-hCT preparations had the greatest hypocalcemic effect. Thus, to enhance the hypocalcemic effect of hCT, large vesicular size is important for I.V. administration and the absence of cholesterol in the liposome is important for I.M. administration. In general, the greatest hypocalcemic effect was achieved by those liposomal preparations that resulted in the most sustained increase of L-hCT and F-hCT in plasma. Thus, liposomal entrapment enhances the hypocalcemic effect of hCT by prolonging the presence of the peptide in the peripheral circulation.     

Modulation by calcitonin of magnesium and calcium urinary excretion in the rat

We evaluated the effects of human calcitonin (hCT) on electrolyte excretion in hormone-deprived rats, that is, in the absence of endogenous parathyroid hormone, antidiuretic hormone, thyrocalcitonin and glucagon, the effects of which might have interfered with those of exogenous calcitonin. Plasma hCT levels, measured by radioimmunoassay, varied from 0 to 32 ng/ml. In these rats, hCT decreased magnesium (Mg) and calcium (Ca) excretion in a dose-dependent fashion. Maximal decreases were observed for hCT plasma concentrations comprised between 3 and 5 ng/ml, and persisted at the highest doses. Sodium, potassium, water, and total solute excretions were constant in the calcitonin concentration range explored. The same was observed for phosphate, except that slight but significant phosphaturia was elicited by the highest doses. Calcium and phosphate infusions to attenuate the fall in plasma Ca and phosphate concentration subsequent to hCT infusion, did not alter the hormonal effect on Ca and Mg excretion. hCT can therefore directly modulate Mg and Ca reabsorption by the kidney at plasma concentrations within the physiological range. The maximal effects on Mg and Ca reabsorption were obtained at plasma concentrations which are generally reached after maximal stimulation of endogenous calcitonin secretion. It is suggested that in rats, endogenous secretion of calcitonin stimulates Ca and Mg renal reabsorption without modification of sodium and phosphate excretion.     

Hypocalcemic effect of salmon calcitonin following single and repeated nasal and intravenous administration in young rabbits

The effect of the polypeptide salmon calcitonin (sCT) on serum calcium concentrations following intranasal and intravenous administration was studied in young rabbits. A small, hypocalcemic effect was observed after nasal administration of sCT without additives, indicating that the nasal sCT absorption was low. The absorption could be improved by addition of an absorption-enhancing adjuvant to the nasal preparation. The absorption, however, was still far from complete as was apparent from the much stronger effect of intravenously injected sCT. When a number of sCT doses were given during a 10-week period, the hypocalcemic effect per sCT dose in the young rabbits decreased after intravenous and, although less pronounced, after nasal administration. The decreased response to sCT is probably not related to the induction of neutralizing antibodies or desensitization of sCT receptors, but is more likely associated with the age-dependent level of bone activity.     

Hypocalcemic actions of amylin amide in humans.

Amylin (also known as islet amyloid polypeptide and diabetes-associated peptide) has recently been shown by us to have a potent hypocalcemic effect in rat and rabbit owing to inhibition of osteoclast-mediated bone resorption. The hypocalcemic potency of amylin was found to be second only to that of calcitonin (CT) and is 100-fold more potent than calcitonin gene-related peptide. Here we demonstrate that amylin has a hypocalcemic effect in patients with Paget's disease of bone. Both human CT (hCT) and amylin induced a maximum hypocalcemic effect 2 h following intravenous administration of the peptides (p less than 0.001). Although on a molar basis amylin is less potent than CT, it exhibits a significantly prolonged hypocalcemic effect compared to hCT. Here we demonstrate for the first time a profound hypocalcemic effect of amylin in the human, despite sharing only 15% amino acid sequence identity with hCT.     

Chondroprotective action of salmon calcitonin in experimental arthropathies

Summary To assess whether calcitonin exerts an influence on cartilage, three models of arthropathies in rabbits—representing three different modes of cartilage destruction—were used: (1) corticosteroid administration (endocrinological disturbances model): (2) meniscectomy (mechanical stress model); and (3) immobilization of the hind leg (nutritional disorder model). After 12 weeks of methylprednisolone (MP) administration, the rabbit femur heads displayed cartilage erosions, marked decrease of glycosaminglycans (GAG) content, and narrowing of joint spaces. Elevation of serum uronic acid, activity of alkaline phosphatase, and calmodulin content was evident. All these changes were minimal—close to normal—in the group treated for 12 weeks with MP + salmon calcitonin (sCT). Partial meniscectomy and hind leg immobilization caused statistically significant loss of GAG from the cartilage and narrowing of the knee joint space during the same experimental period, 12 weeks. In both these models the groups of rabbits treated simultaneously with sCT showed only insignificantly smaller joint spaces and GAG content. These results support our hypothesis of a chondroprotective property of calcitonin. However, the mechanism through which calcitonin influences joint cartilage remains unknown. A direct effect of calcitonin on cultivated chondrocytes, as well as the role of calmodulin, beta-endorphins, calcium, and interleukin-1 in the process are discussed.     

Stimulatory effect of insulin-like growth factor binding protein-5 on mouse osteoclast formation and osteoclastic bone-resorbing activity.

Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates osteoblast proliferation directly or indirectly through IGF-I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP-5 on osteoclastic activity and osteoclast formation. IGFBP-5 significantly stimulated pit formation by pre-existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF-I antibody (Ab). However, IGFBP-5 did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When IGFBP-5 was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, IGFBP-5 (77 pM-7.7 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of IGF-I Ab. Moreover, osteoclast-like cells newly formed by IGFBP-5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP-5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP-5 dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors, irrespective of the presence of IGF-I Ab. Growth hormone (GH) as well as IGF-I significantly stimulated bone resorption by pre-existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF-I Ab. GH as well as IGF-I stimulated osteoclast-like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF-I Ab. The direct stimulatory effect of GH on osteoclast-like cell formation from hemopoietic blast cells was not affected by IGF-I Ab. The present data indicate that IGFBP-5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF-I-independent fashion and by IGF-I-dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro.     

Sustained release of salmon calcitonin in vivo from lactide: Glycolide copolymer depots

Summary Studies were carried out to determine whether monolithic depot formulations, prepared using lactide:glycolide copolymers, could be used to administer salmon calcitonin (sCT) to rats in vivo. Formulations containing 2, 5, or 10% (w/w) sCT were administered subcutaneously to female Wistar strain rats. Release of sCT was determined by measurement of peptide in plasma using a specific radioimmunoassay and by measurement of residual sCT in the depots after recovery at postmortem. Plasma calcium concentrations and cumulative weight gain of the animals were used to measure pharmacological effects of the released sCT. Release of sCT from the depots was controlled by the copolymer and was sustained for periods up to 10 days. However, the release of sCT from the depots did not significantly alter plasma calcium concentrations, and effects on cumulative weight gain were small and transient. Peptide loading of the formulations was shown to modify sCT release. Maximal release of sCT from depots containing 10% peptide occurred over a 7 to 14-day period postadministration, with 5% sCT release occurred between days 11 and 14, and with 2% sCT, the period of maximal release was between days 11 and 18. Release of peptide from the depots was essentially complete by 21 days postadministration irrespective of the peptide loading. These data suggest that lactide:glycolide copolymer depots may have application for the convenient clinical administration of sCT in metabolic bone diseases.     

Nasal salmon calcitonin in osteoporosis

Conclusion Nasal sCT is a safe drug with few and mild side effects and compared with HRT is often more attractive to elderly, osteoporotic women. Under controlled conditions, nasal sCT reduces the rate of new fractures by two-thirds compared with calcium treatment alone [10] and increases spinal bone mass dose dependently.Conclusively, nasal sCT is indicated for treating women who have passed the menopause some years ago. The treatment should be administered discontinuously. The nasal formulation has been approved for treatment of osteoporosis in a variety of European and South American countries, and has also been filed with appropriate governmental authorities for consideration of approval in many countries throughout the world, including the US. Should calcitonin treatment be generalized in women who are 10–15 years postmenopausal, the costs will undoubtedly be reduced. Compared with the effect of HRT on early postmenopausal bone loss, the effect of nasal sCT is only marginal. Thus, HRT prevents both the cortical and trabecular bone loss throughout the entire treatment period [38]. Furthermore, HRT is inexpensive and its adverse event profile has been fully clarified except in respect to the possible change in the risk of breast cancer [39]. Nasal sCT can be effective in women soon after the menopause—by decreasing the rate of vertebral bone loss and lowering the bone turnover—as an alternative to hormone replacement therapy either when estrogens are contraindicated or for women who are not candidates for estrogen therapy.     

Identification of biphenylcarboxylic acid derivatives as a novel class of bone resorption inhibitors.

A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy-induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs. INTRODUCTION: Many common bone diseases such as osteoporosis, Paget's disease, and cancer-associated bone disease are characterized by excessive bone loss caused by increased osteoclastic activity. Successful treatment of these diseases is based on osteoclast inhibition. The osteoclast inhibitory drugs that are currently available fall into relatively few mechanistic classes, indicating the need to identify novel antiresorptives. Here we describe a series of biphenylcarboxylic acid derivatives that have potent inhibitory effects on osteoclastic bone resorption in vitro and on ovariectomy-induced bone loss in vivo. MATERIALS AND METHODS: Compounds were tested for inhibitory effects on bone resorption in vitro using mouse osteoblast-bone marrow co-cultures, isolated rabbit osteoclasts, and mouse osteoclasts generated from bone marrow. Some experiments were also performed on human osteoclasts generated from peripheral blood mononuclear cells. We also investigated the effects of specific compounds on ovariectomy-induced bone loss in vivo in mice. RESULTS: One of the most potent compounds identified was the butanediol ester of biphenyl carboxylic acid (ABD056), which inhibited osteoclast formation in mouse osteoblast-bone marrow co-cultures by 50% (IC50) at a concentration of 26 microM and in macrophage-colony stimulating factor (M-CSF)- and RANKL-stimulated mouse bone marrow cultures with an IC50 of 8 microM. Mechanistic studies showed that ABD056 caused osteoclast apoptosis and inhibited TNFalpha-induced NF-kappaB activation. No inhibitory effects on osteoblast growth or differentiation were observed at concentrations of up to 100 microM. When administered to mice at doses of 5 and 10 mg/kg/day, ABD056 prevented ovariectomy-induced bone loss. CONCLUSIONS: Butanediol biphenylcarboxylic acid derivatives represent a new class of antiresorptive drug that might be of therapeutic value in the prevention and treatment of diseases characterized by osteoclast activation such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone.     

Oxygen derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction

Oxygen derived free radicals are generated by osteoclasts. In a novel culture system, isolated rat osteoclasts were stained when nitroblue tetrazolium (NBT) was reduced by cellular oxidants to formazan, an insoluble precipitate. Superoxide dismutase (SOD) inhibited the accumulation of formazan by the isolated osteoclasts. Osteoclasts in mouse calvarial organ cultures also reduced NBT to formazan. The reaction products were localized to the area of the osteoclast-bone interface. At the light microscopic level, the formazan granules appeared to be concentrated within the cytoplasm. Formazan accumulation was significantly inhibited by calcitonin (hCT). The inhibition of NBT reduction by SOD indicates that the isolated osteoclasts were capable of producing superoxide. The localization of the formazan granules between the external osteoclastic membrane and the bone, and the inhibition of this reaction during hCT exposure suggests that oxygen derived free radicals may contribute to bone resorption.     

RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin.

The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action.     

Intracerebroventricular calcitonin prevents stress-induced gastric dysfunction

BACKGROUND: Restraint stress produces gastric hypercontractility and acidity leading to stress ulceration. Intracerebroventricular (ICV) salmon calcitonin (sCT) decreases restraint injury and acidity, but its effects on restraint-induced hypercontractility are unknown. METHODS: Using stereotactic guidance, ICV catheters were placed into the lateral ventricle of adult male rats and calibrated gastric strain gauge transducers were implanted 5 days prior to restraint stress. sCT rats (n = 8) were pretreated with 5 microg of calcitonin ICV (10 microl volume), while controls (n = 10) received 10 microl of ICV saline prior to restraint for 2 h at 20 degrees C followed by 2 h at 4 degrees C. Gastric motility data were collected with AT-CODAS and analyzed with ADVANCED CODAS. Gastric volume, pH, and lesions were recorded following the stress. RESULTS: ICV calcitonin prevented gastric mucosal injury in all animals (0% vs 100%, P <.01) and elevated pH slightly (2.5 +/-.3 vs 1.6 +/-.1, P <.05). Stress caused the force of contractions to increase from 0.35 +/-.1 to 1.38 +/-.4 g in controls (P <.01), while treated animal's force fell from.42 +/-.1 to 0.2 +/-.05 g (P <.01 vs control). Stress did not affect contractions/min (3.4 +.6 vs 3.5 +.3), but sCT increased frequency (2.5 +.4 to 5.0 +.2, P <.01). Stress prolonged contraction duration (11.5 + 1 to 16.5 + 1.7 s, P <.01), but stress's effect was prevented by sCT (11.0 +.5 to 11.2 +.3, P <.01 vs control). CONCLUSIONS: Pretreatment with 5 microg central sCT prevents the increased amplitude and duration of gastric contractions produced by restraint stress for 2 h, in association with gastroprotection.