Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2834-2838, April 1989 Medical Sciences Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach (calcium incorporation/calcium homeostasis/hypocalcemic hormone) PER PERSSON, ROLF HAKANSON*, JAN AXELSON, AND FRANK SUNDLER Departments of Pharmacology, Surgery, and Medical Cell Research, University of Lund, Lund, Sweden Communicated by Jan Waldenstrom, September 19, 1988 (received for review August 4, 1988)

ABSTRACT Gastrin-17 induces hypocalcemia in the rat MATERIALS AND METHODS without stimulating calcitonin release. The gastrin-induced hypocalcemia persisted after thyroparathyroidectomy or para- Animals. Male Sprague-Dawley rats, weighing 150-225 g, thyroidectomy. In contrast, gastrectomy or extirpation of the were used. The rats were fasted overnight (18-24 hr) before acid-producing part of the stomach each experiment. prevented the hypocalce- Enzymes and Synthetic Peptides. Synthetic human gastrin- mic effect, suggesting the involvement ofthe proximal stomach 17 (nonsulfated) and human calcitonin were from Peninsula in the gastrin-evoked lowering of blood calcium. The drop in Laboratories. Fresh solutions were prepared each day. The blood calcium upon i 'ection of gastrin-17 did not reflect a loss two peptides were dissolved in 0.9%o saline and administered of calcium via the gastric juice or via the urine. Extracts of the subcutaneously (s.c.) or into the jugular vein in a total vol of acid-producing mucosa of the rat stomach had a hypocalcemic 1 or 2 ml (s.c.) and 0.2 ml (i.v.). The administration was s.c. effect. The extracts were purified by gel chromatography and unless otherwise stated. Control rats were injected with the reversed-phase high-performance liquid chromatography. Di- appropriate volume of 0.9% saline. Leucine aminopeptidase gestion with leucine aminopeptidase destroyed the hypocalce- (microsomal type IV-S, porcine kidney) and trypsin (type III, mic activity, while trypsin had no effect, suggesting a peptide bovine) were obtained from Sigma. (or peptides) with an unprotected NH2 terminus and without Surgical Procedures. Operations were performed under basic amino acid residues (or with protected basic amino acids). light diethyl ether anesthesia. Parathyroidectomy and com- Both gastrin-17 and the mucosal extract stimulated the uptake bined thyroidectomy and parathyroidectomy were per- of45Ca into bone (radius and sternum). Gastrin-17 was without formed under an operation microscope. The proximal part of effect in rats that had undergone gastrectomy, while the the stomach was resected (fundectomy) as described in detail mucosal extract was equally effective in gastrectomized and elsewhere (5). Gastrectomy was performed by resection of unoperated rats. We suggest that the effects of gastrin-17 on the stomach followed by joining the esophagus and duode- blood calcium and on calcium transfer into bone are indirect num end-to-end. The animals were allowed to recover for 7 and that gastrin-17 stimulates the release ofa peptide hormone, days after the operation. tentatively named gastrocalcin, from the acid-producing mu- Blood Sampling. Rats were placed in Bollman-type re- cosa ofthe stomach. Gastrocalcin stimulates the uptake of45Ca straining cages and injected in the proximal part of the tail into bone, thereby causing hypocalcemia. with lidocain (Xylocain; Astra, Sodertalje, Sweden) 1 hr before the start of the experiment and every hour thereafter. Calcium plays a major role in fundamental biological pro- Blood was drawn from the tip of the tail. Each rat was cesses, such as bone muscle and sampled at least four times. mineralization, contraction, Ca in Blood. The effect of s.c. or i.v. injection ofgastrin-17 intracellular signal transduction. In vertebrates the control of or s.c. injection of an extract of acid-producing (oxyntic) Ca2+ in blood involves the parathyroid hormone, calcitonin, mucosa (for details on the preparation of the extract, see and vitamin D. The parathyroid hormone stimulates the below) was studied by measuring the levels of circulating release of Ca2l from bone, the reabsorption of Ca2l in renal calcium before and 20, 60, and 180 min after injection. For tubules, and the conversion ofinactive 25-hydroxyvitamin D determination of ionized calcium, 150 ,u offresh whole blood to 1,25-dihydroxyvitamin D, which then enhances the ab- was collected in heparinized capillary tubes and analyzed at sorption of Ca2+ from the intestines. The effects of parathy- once using a Ca2+ selective electrode (ICA1; Radiometer, roid hormone are reflected in hypercalcemia. Calcitonin, Copenhagen). The pH ofthe blood was measured by the same which is produced in the C cells ofthe thyroid, suppresses the electrode. Ifthe pH was below 7.36 the samples were not taken release of Ca2+ from bone and causes hypocalcemia. into account. Total calcium was determined spectrophotomet- In 1975, Schulak and Kaplan (1) reported that porcine rically in 25 ,ul of serum with a commercially available kit gastrin evokes hypocalcemia in intact as well as in thyropar- based on the conjugation of calcium with o-cresolphthalein athyroidectomized rats, suggesting a mechanism not involv- (Calcium C; Wako Chemicals, Neuss, F.R.G.). ing calcitonin. This view is supported by the fact that in the Ca in Gastric Juice and Urine. Fistulas were generated by rat neither exogenous gastrin-17 nor hypergastrinemia of implantation of plastic tubes in the rumen of the stomach endogenous origin stimulated calcitonin release (cf. ref. 2). under diethyl ether anesthesia. The rats were allowed to Gastrectomy or resection of the proximal stomach inhibited recover for 2 weeks. Before the start of each experiment, the the hypocalcemic response to gastrin (1, 3, 4), indicating that gastric fistula was opened, and the stomach was rinsed with the proximal stomach mediated the response. The present warm saline and allowed to drain freely for 1 hr. Gastrin-17 study examines the mechanism by which gastrin induces or saline was injected and gastric juice was collected in hypocalcemia and the role played by the proximal stomach. 30-min portions. The volumes were measured, the acid output was determined by titration with 0.02 M NaOH, and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: Department of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Pharmacology, Solvegatan 10, 223 62 Lund, Sweden. 2834 Downloaded by guest on September 24, 2021 Medical Sciences: Persson et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2835 the total calcium output was determined spectrophotometri- Table 1. Basal circulating calcium levels in fasted unoperated cally with 50 ul of gastric juice. Plastic fistulas were im- and operated rats planted in the urinary bladder under diethyl ether anesthesia Ca2+ in Total Ca and the rats were allowed to recover for 3 days. Before the whole blood, in serum, start of each experiment, the fistula was opened, and the Operation mM mM bladder was rinsed with warm saline and allowed to drain freely for 1 hr. Gastrin-17 or saline was injected and urine was No operation 1.29 ± 0.01 (10) 2.34 ± 0.03 (8) collected in 15-min portions. The volumes were measured, Gastrectomy 1.28 ± 0.02 (5) 2.33 ± 0.03 (3) and the total calcium output was determined by the spectro- Fundectomy 1.28 ± 0.01 (3) 2.35 ± 0.03 (5) photometric method with 50 ,ul of urine. Parathyroidectomy 0.71 ± 0.01 (5) 1.14 ± 0.04 (5) Uptake of Peroral 45Ca in Blood. 45CaC12 (5 ,Ci per kg of Thyroparathyroidectomy 0.69 ± 0.01 (6) 1.09 ± 0.02 (6) body weight; specific activity, 4-50 Ci/g; 1 Ci = 37 GBq; Gastrectomy + NEN-Chemicals, DuPont, Dreieich, F.R.G.) was adminis- thyroparathyroidectomy 0.67 ± 0.02 (6) tered in 1 ml of0.9%o saline orally via a gastric tube. Injection Levels were measured 1 week after the operations. Results are ofgastrin and sampling ofblood was made 15 min later. Blood expressed as means ± SEM. Numbers in parentheses represent samples (50 ,ul) were then collected at various time intervals number of rats. in capillary tubes and added to an aliquot of solubilizer [150 ,ul of Soluene-350/isopropanol (1:2)]. The mixture was incu- extract, see below.) The radius and sternum were dissected bated for 1 hr at 40°C. Decolorization was achieved by adding out, cleaned, and placed in an oven at 800'C for 24 hr. The 200 ,ul of 30% hydrogen peroxide and leaving the mixture for resulting ash was weighed and dissolved in 1 M HCl. Picofluor- 2 hr at room temperature (6). After decolorization, 10 ml of 40 (10 ml) was added. The radioactivity of the samples was Picofluor-40 (Packard Instrument) cocktail was added to each counted after overnight equilibration. sample. The vials were capped, vortex mixed, and left Preparation of Oxyntic Mucosal Extract. Altogether, six standing overnight at room temperature before determination preparations were made; 100-120 rats were killed for each of radioactivity (LKB p-counter Wallac 1214 Rackbeta). preparation. The stomachs were dissected out and opened. Also, 45Ca in deproteinized blood was analyzed; 50 ,ul of The antrum was cut away. The mucosa was scraped off the blood was added to 100 ,l of 5% trichloroacetic acid in oxyntic gland area with a scalpel, weighed (the wet weight of microcentrifuge tubes. The samples were centrifuged for 10 the pooled material varied between 70 and 80 g), and homog- min at 1000 x g. A 10-ml Picofluor-40 cocktail was added to enized in ice-cold acetone (10 ml per g of tissue). The each supernatant, and the radioactivity of the deproteinized sediment was collected, washed with ice-cold acetone, fol- samples was determined after overnight equilibration. lowed by acetone-diethyl ether (50:50), and diethyl ether. The Uptake of45Ca into Bone. For the study of45Ca transfer into sediment was then taken up in 500 ml of 1 M acetic acid, bone, the rats were killed by an overdose of diethyl ether 55 boiled for 30 min, and left overnight. Undissolved material min after the peroral or s.c. administration of 45Ca. The rats was sedimented by centrifugation at 6000 x g at +40C for 20 had received gastrin-17 (usually 100 ,ug/kg) or oxyntic mucosal min. The supernatants were freeze-dried and taken up in 0.2 extract (1 mg per kg ofbody weight in 1 ml of0.9%o saline s.c.) M acetic acid, boiled for 30 min, and left standing overnight. 40 min before sacrifice. (For details on the preparation of the Precipitated material was sedimented by centrifugation at &Ca2+ A fACa2+ C mmol/l min x mmol/l 0.021 2.0 No NS NS operation (10) NS FX (3) 0 0. GX (5) ('9 ()7(88 PTX (5) (7) (7) (8) THX (6) THX+bX (6) -0.02 -2.

O No operation Saline -0.04 * No operation} Gastrin-17 O3 FX I -4.0 0 20 60 160 min FX PTX THX+GX ACatot No B operation GX THX mmol/l Saline [- Gastrin-17 0.10 f Ca tot min x mmol/l 10.0] No operation (8) D O yA (IM 15) (5) D 0. GX (5) PTX (5) -0. 10 THX (6) I

-0.20

CD 20 60 180 min -30.0 FIG. 1. Influence of gastrectomy (GX), fundectomy (FX), parathyroidectomy (PTX), thyroparathyroidectomy (THX), and THX+GX on the effect of gastrin-17 (100 ,ug/kg) on whole blood ionized calcium (Ca2+) (A and C) and serum total calcium (Cat~t) (B and D) in fasted rats. Operations were 1 week before the experiment. Gastrin-17 or saline was injected s.c. at arrow. Results are expressed as means ± SEM. Basal calcium levels are given in Table 1. (A and B) Time course ofthe hypocalcemic response (expressed as change over starting value) to gastrin-17. Fundectomy prevented the effect. (C and D) Integrated blood calcium changes after injection of gastrin-17. Calculations are made on results such as those shown in A and B (*, P < 0.05; **, P < 0.01 by Student's t test). NS, not significant. Numbers in parentheses represent number of rats. Downloaded by guest on September 24, 2021 2836 Medical Sciences: Persson et al. Proc. Nad. Acad. Sci. USA 86 (1989) 25,000 x g for 15 min. The supernatant was freeze-dried, with 0.1 M NH4CO3 buffer (pH 8.0) for 16 hr. Controls weighed, and taken up in 0.2 M acetic acid. At this stage, the included material incubated without enzyme and enzyme weight of the freeze-dried material was 5-6 g. Portions of without hypocalcemic material. The incubation mixtures 2.5-3 g in 10-20 ml were passed through a Sephadex G-50fine were injected s.c. (four rats in each group). column (90 x 2.6 cm), equilibrated, and developed with 0.2 M acetic acid. Proteins/peptides were monitored (7) with bovine serum albumin (United States Biochemical) used as RESULTS AND COMMENTS standard. Fractions of 2-3 ml were collected, freeze-dried, Effect of Surgery on Blood Calcium. One week after gas- and weighed; 100-200 .ug of each fraction was reconstituted trectomy or fundectomy, the blood calcium levels were in 0.2 ml of 0.9% saline and tested for calcium-lowering activity after i.v. injection. The fractions containing such Sephadex G-50 activity were pooled and purified further by repeated re- Met-Enkephalin HPLC using a puBondapak C18 column (7.8 x mg protein IBlue Dextran 3H20F versed-phase /ml 300 mm). The solvent was 0.08% trifluoroacetic acid with 0- CRF Coloured I 50% acetonitrile (as specified). The flow rate was 1 ml/min. 10 f~ In the first, but not in the subsequent, HPLC steps the column was overloaded. The overloading of the column was reflected in a >10% change in the elution volume for the 5' fractions containing hypocalcemic activity. The HPLC sys- tem consisted of a Waters model 680 automated gradient controller equipped with a model U6K injector, a 6000 pump, 100 200 300 400 and a model 440 absorbance detector (214 nm). Fractions of 0.5 ml were collected and freeze-dried. The dry residues were redissolved in 0.9% saline for assessment of calcium- lowering activity. In one series of experiments, fractions from the Sephadex G-50 column, containing calcium-lowering activity, were collected, freeze-dried, and subjected to digestion with tryp- sin or leucine aminopeptidase. Trypsin (5 tug; 50 units) was dissolved in 600 ttl of0.1 M Tris buffer (pH 8.0) and incubated with 200 ,ug of the freeze-dried material for 6 hr at 370C. For control purposes, 200 pug ofthe material was incubated under identical conditions but without trypsin, and trypsin was incubated without freeze-dried material. The digestion was terminated by boiling for 1 hr. Digestion with leucine ami- nopeptidase (10 units) was carried out as described above,

Gastrin-17 big/kg body weight 12.5 25 50 100 200 0- A

-0.02- 40 60 20 Retention Time (min) FIG. 3. Gel chromatography of an extract of rat oxyntic mucosa. Freeze-dried material (2.5 g), prepared from 35 g of oxyntic mucosa -0.04- (see text for details), was dissolved in 20 ml of 0.2 M acetic acid and passed through a Sephadex G-50 fine column. The column was developed with 0.2 M acetic acid. Fractions (3 ml) were collected and A ca22 freeze-dried. The drawn curve represents protein/peptides. The rnmol/l column was calibrated with blue dextran (void volume) and 3H20 (total volume). The elution volumes ofcorticotropin-releasing factor 12.5 25 50 100 200 (CRF) and [Met]enkephalin are indicated. The hatched areas repre- 0. B sent material with hypocalcemic activity (tested as described in Fig. 4). Each lyophilized fraction was dissolved in 1 ml of 0.9% saline; 100-200 ,u was injected i.v. Blood Ca responses were monitored. -0. 10. The pooled hypocalcemic material weighed 300 mg after lyophiliza- tion and was purified by reversed-phase HPLC (for details, see text). Eight milligrams was applied in each run; this overloaded the -0. 20- column. The column was developed with an isocratic elution using 0.08% trifluoroacetic acid and 10%1 acetonitrile for 40 min. followed by a linear gradient of 10-50%o acetonitrile for 30 min. Fractions of 0.5 ml were collected and freeze-dried. Two zones with hypocalce- -0.301 fraction for i.v. A Catot mic activity were found using one-fifth of each mmol/l injection. The two zones were repurified by HPLC. Elution was with 0.08% trifluoroacetic acid and a linear gradient of 0-16.5% acetoni- FIG. 2. Dose-response curves illustrating the hypocalcemic trile for 60 min. Fractions of 0.5 ml were collected, freeze-dried, and effect (expressed as change over starting value 20 min after injection) tested for hypocalcemic activity with one-tenth of each fraction for of s.c. injection of gastrin-17. Whole blood ionized calcium (Ca2+) in i.v. injection. The purification procedure was applied on six prepa- A and serum total calcium (Cat~t) in B. Each value is the mean of six rations, each starting with 70-100 g of mucosa. The results presented determinations. Vertical bars give SEM. are from a typical run. Downloaded by guest on September 24, 2021 Medical Sciences: Persson et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2837 unchanged. Parathyroidectomy and thyroparathyroidec- Effect of Gastrin-17 and Oxyntic Mucosal Extract on Blood tomy, on the other hand, lowered the blood calcium levels 45Ca. In rats given 45Ca orally, gastrin-17 and the oxyntic (Table 1). mucosal extract lowered the concentration of 45Ca in both Hypocalcemic Effect of Gastrin-17. Gastrin-17 induced hy- whole blood and deproteinized blood (Fig. 5). pocalcemia in unoperated, thyroparathyroidectomized, and Effect of Gastrin-17 and Oxyntic Mucosal Extract on the parathyroidectomized rats but not in gastrectomized or fun- Uptake of45Ca into Bone. Gastrin-17 enhanced the deposition dectomized rats (Fig. 1). The drop in total calcium was of perorally administered 45Ca in the radius and sternum of greater than that in ionized calcium and more long-lasting; the control (Fig. 6A) and thyroparathyroidectomized rats (data responses for total calcium and for ionized calcium peaked 60 not shown) but not of gastrectomized rats (Fig. 6B). The and 20 min, respectively, after the injection ofgastrin-17. The effect was dose dependent (Fig. 7). The results were identical hypocalcemic response was dose dependent (Fig. 2). Intra- when 45Ca was given s.c. (data not shown). Calcitonin (1 venous administration of gastrin-17 produced a 60% greater ,ug/kg s.c.) produced marked hypocalcemia but was ineffec- peak response than subcutaneous injection; the time courses tual on calcium transfer into bone (data not shown). The of the responses were identical (data not shown). oxyntic mucosal extract enhanced the uptake of 45Ca into Effect of Gastrin-17 on Calcium in Gastric Juice and Urine. bone in both unoperated (Fig. 6A) and gastrectomized rats Gastrin-17 (100 ,ug/kg s.c.) produced a marked acid response (Fig. 6B). in the five fistula rats studied but the stimulated acid secretion was not accompanied by an increased output ofcalcium in the gastricjuice and was not accompanied by an increased output DISCUSSION of calcium in the urine (data not shown). Five control rats Gastrin produces hypocalcemia in the rat. Neither exogenous received 0.9% saline. nor endogenous gastrins seem to stimulate the release of Hypocalcemic Effect of Oxyntic Mucosal Extract. An acetic calcitonin in the rat (2), and the hypocalcemic response acid extract, prepared from oxyntic mucosa, was passed evoked by gastrin-17 must therefore have another explana- through a Sephadex G-50 column and the fractions were tion. Schulak and Kaplan (1) and Kaplan et al. (3) were first analyzed for hypocalcemic activity (Figs. 3 and 4). Digestion to report that thyroparathyroidectomy and parathyroidec- with leucine aminopeptidase degraded the hypocalcemic tomy had no effect on the gastrin-evoked hypocalcemia while activity but trypsin was without effect (data not shown). The gastrectomy or fundectomy prevented it. Krishnamra and fractions that contained hypocalcemic activity were pooled Limlomwongse (8) and Limlomwongse and Krishnamra (9) and purified further by repeated HPLC. At least two separate suggested that neither gastric secretion of calcium nor renal peaks displayed hypocalcemic activity (Fig. 3). Percent of Whole blood A ACa2+ control mmol/l A 110- 0.021

0 -6 (6) 100 'r ~~~~~~~~~~~(6) -0.02- 90 -0.04 - I o Saline -0.06- * Mucosal extract 80 -0R.08 - * Gastrin-17 0 20 60 180 4 O Mucosal extract min o 20 60 180 min ACa 2+ pg/kg body weight Percent of mmol/l control I B 50 100 200 400 E800 1600 1101 Deproteinized blood 0 1 B 1(4) 100 -0.02 |(7) 90o

-0.04 80

-0. 06- 701 4 FIG. 4. (A) Time course of the hypocalcemia (change over 0 20 60 180 min starting value) evoked by s.c. injection (arrow) of 0.9o saline or of an extract of the acid-producing mucosa. The dose given was 800 FIG. 5. Time course ofthe effects ofgastrin-17 orgastric mucosal ,ig/kg, expressed as the weight ofthe freeze-dried biologically active extract on whole blood 45Ca (A) and on 45Ca in deproteinized blood material collected from the Sephadex G-50 column. One milligram of (B) in fasted rats. The rats received 45Ca perorally 15 min before this material corresponds to -230 mg fresh mucosa. Results are sampling. The gastrin dose was 100 ,ug/kg and the amount ofextract expressed as means ± SEM. Numbers in parentheses refer to the injected corresponded to 1 mg per kg of freeze-dried material from number of rats. (B) Dose-response curve showing the hypocalcemic the Sephadex G-50 column (hatched area in Fig. 3). Injections were response (expressed as change over starting value 20 min after given s.c. at arrow. Results are expressed in relation to the mean injection) to mucosal extract. Doses given are ,ug of the material values in the same number of control rats receiving 0.9o saline described above. Each value is the mean of three to eight determi- instead of gastrin-17 (seven rats) or mucosal extract (four rats) nations. (percent change ± SEM). Downloaded by guest on September 24, 2021 2838 Medical Sciences: Persson et al. Prot. Nad. Acad. Sci. USA 86 (1989)

No operation A cpm/mg ash Sternum 4001 Sternum

300' F--'

200 I,- ,R Gastrectomy B cpm/mg ash Sternum

100

r - - - -.i 0.9% 12.5 25 50 100 200 NaCl Gastrin-17 (pg/kg body weight) FIG. 7. Dose-response curves illustrating the effects of gastrin- FIG. 6. (A) Effects of gastrin-17 (100 ,ug/kg s.c.) or a mucosal 17 on the uptake of 45Ca into radius and sternum. P values for the extract (1 mg/kg s.c. offreeze-dried material from the Sephadex G-50 regression coefficients are as follows: radius, <0.05; sternum, <0.01. column) on the uptake of 45Ca into bone (radius and sternum) of Each value is the mean of five determinations. Vertical bars give unoperated rats. 45CaCI2 was given orally 15 min before injection of SEM. gastrin-17 or extract and 55 min before sacrifice. The incorporation of An extract of oxyntic mucosa, purified combined 45Ca was enhanced by gastrin-17 and by the mucosal extract. (B) by gel Gastrectomy prevented the enhancing effect of gastrin-17 on the chromatography and reversed-phase HPLC, could be shown incorporation of 45Ca into bone, but not that of the mucosal extract. to mimic the effects of gastrin-17 on blood calcium and on (*, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t test.) NS, not calcium uptake into bone. The chromatographic behavior of significant. Numbers in parentheses represent number of rats. the oxyntic mucosal agent and the loss ofhypocalcemic effect on digestion with leucine aminopeptidase suggest that it is a elimination of calcium the small peptide with an unprotected NH2 terminus. Its resist- could explain gastrin-evoked hy- ance to trypsin digestion suggests a lack of basic amino acid pocalcemia. They also reported that gastrin suppressed efflux residues (or protected basic amino acids). We propose the of calcium from bone (9) and proposed that gastrin exerted a name gastrocalcin for this putative peptide hormone, which direct action on bone calcium. Our results suggest that the is produced in the oxyntic mucosa and released by gastrin. It hypocalcemic response to gastrin-17 is indirect, involving the accelerates calcium uptake into bone, thereby explaining the release of hypocalcemic agents from the oxyntic mucosa of hypocalcemia evoked by gastrin. To the three functionally the stomach. The gastrin-induced hypocalcemia reflected interacting factors controlling Ca2+ concentration in blood neither the loss of calcium in the gastric juice nor the loss of (parathyroid hormone, calcitonin, and vitamin D), we wish to calcium in the urine. Also, it appeared unlikely that the add a fourth-namely, gastrocalcin. gastrin-induced hypocalcemia reflected an impaired uptake of calcium from the intestines for the simple reason that the This work was supported by the Swedish Medical Research Council (04X-1007), AB Hassle, and the Tercentenary Foundation of rats were fasted before the experiments and, consequently, the Bank of Sweden. not much calcium could be absorbed from the intestines under the circumstances. In fact, one series of experiments 1. Schulak, J. A. & Kaplan, E. L. (1975) Endocrinology 96, 1217- was run in which gastrin-17 induced the same degree of 1220. hypocalcemia after prolonged fasting (48 hr) as after over- 2. Persson, P., Grunditz, T., Axelson, J., Sundler, F. & Hakan- night fasting (data not shown). Finally, we could show that son, R. (1988) Regul. Pept. 21, 45-56. gastrin-17 enhanced the uptake of45Ca into bone and 3. Kaplan, E. L., North, P. T., Norberg, H. P., Schulak, J. A. & (radius Hill, B. J. (1977) J. Surg. Res. 22, 237-241. sternum), which could account for the gastrin-17-evoked 4. Klementschitsch, P., Kaplan, E. L., Heath, H., North, P. & hypocalcemia. Conceivably, gastrin might act directly on Lee, C.-H. (1979) Endocrinology 105, 1243-1247. bone or indirectly by releasing an agent that enhanced 5. Alumets, J., El Munshid, H. A., Hakanson, R., Hedenbro, J., calcium transfer. An indirect action was in fact suggested by Liedberg, G., Oscarson, J., Rehfeld, J. F., Sundler, F. & the finding that gastrin-17 was without effect on calcium Vallgren, S. (1980) J. Physiol. (London) 298, 557-569. uptake into bone in gastrectomized rats, whereas extracts of 6. Moore, P. A. (1981) Clin. Chem. 27, 609-611. oxynic mucosa stimulated calcium transfer well in 7. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. equally (1951) J. Biol. Chem. 193, 265-275. unoperated and gastrectomized rats. These results may be 8. Krishnamra, N. & Limlomwongse, L. (1978) Proc. Soc. Exp. interpreted to mean that the hypothetical hypocalcemic agent Biol. Med. 158, 40-44. that is released by gastrin-17 emanates from the oxyntic 9. Limlomwongse, L. & Krishnamra, N. (1981) Proc. Soc. Exp. mucosa of the stomach. Biol. Med. 168, 72-76. Downloaded by guest on September 24, 2021