Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatin- like immunoreactivity (SLI) and cytosolic binding sites for ...
Bioscience Reports, Vol. 8, No. 3, 1988
Effects of Alloxan-Induced Diabetes on Somatostatin Binding to Cytosolic Components of Rabbit Gastric Fundic Mucosa L. G. Guijarro, M. P. Lopez-Ruiz, J. C. Prieto and E. Arilla 1 Received February 15, 1988 Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatinlike immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyral-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma. KEY WORDS: somatostatin binding; gastric mucosa; alloxan; diabetes.
INTRODUCTION Somatostatin is a widely distributed peptide in the gastrointestinal tract (1, 2) which may act locally as a paracrine substance (3). Experimental evidence exists for the release of somatostatin into the lumen of the gastrointestinal tract and for the secretion of splanchnic somatostatin into the circulatory system following the ingestion of a meal (4, 5). In addition, receptors for somatostatin have been characterized in the gastrointestinal tract (6-9). In diabetes mellitus associated with insulin-deficiency, an increase of gastric somatostatin content has been demonstrated in rats (10, 11). However, the regulation of intracellular somatostatin binding sites in diabetic animals has not been studied. Therefore, the present investigation was undertaken to determine the; effects of alloxan-induced diabetes (three months after the onset of the Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Alcala de Henares, Madrid, Spain. 1 To whom correspondence should be addressed. 233 0144-8463/88/0600-0233506.00/0 (~) 1988 Plenum Publishing Corporation
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disease) on specific somatostatin binding sites in rabbit gastric fundic mucosa. The study also included the determination of SLI concentrations in fundic mucosa and plasma from portal vein, aorta and vena cava.
MATERIALS AND METHODS Somatostatin-14 was provided by Serono (Madrid, Spain); synthetic Tyr nsomatostatin was purchased from Universal Biologicals Ltd. (Cambridge, UK); trypsin inhibitor and bovine serum albumin from Sigma (St Louis, MO, USA); and Na 1251 (100mCi/ml) from Radiochemical Centre (Amersham, UK). Synthetic Tyrn-somatostatin was radioiodinated at a specific activity of about 360 Ci/g (12). The rabbit antibody used in the radioimmunoassay technique was purchased from the Radiochemical Centre (Amersham, UK). This antiserum was raised in rabbits against somatostastin-14 conjugated to bovine serum albumin and is specific for somatostatin but, as somatostatin-14 constitutes the C-terminal portions of both somatostatin-25 and somatostatin-28, the antiserum does not distinguish between these three forms. All other chemicals were reagent grade. The study was carried out on New Zealand white male rabbits weighing 1-2 Kg. After 24-hour fasting, animals received a single intravenous injection of alloxan (150mg/kg) dissolved in sterile distilled water (13). Control animals received saline alone. In order to confirm the development of diabetes, each rabbit was bled by ear puncture 7 days following alloxan administration, plasma being separated and frozen at -30~ until assay. Plasma insulin concentration was measured by radioimmunoassay (14). Plasma glucose was assayed by the glucose-oxidase (Boehringer) method. Three months after alloxan-induced diabetes, the rabbits were anesthetized with ketamine hydrochloride and laparotomized. Blood samples were collected from the portal vein, aorta and inferior vena cava in chilled tubes containing 1000 KIU/ml aprotinin and 1.2 mg/ml EDTA and immediately centrifuged, plasma being stored at -30~ until assay. The animals were then killed by cervical dislocation, the gastric fundus being immediately removed. Radioimmunoassay of Somatostatin Somatostatin immunoreactivity in gastric fundic mucosa was extracted in 1M acetic acid at 0~ by sonication. Peptide concentration was determined in both plasma and tissue extracts by a radioimmunoassay method (15). 12SI-Tyr11somatostatin was used as tracer together with a highly specific rabbit antisomatostatin serum (initial dilution 1:20,000) which gives a lower limit of detection of 10 pg/ml. Samples were run in triplicate. Separation of bound and free hormone was accomplished with dextran-coated charcoal. Dilution curves for plasma and tissue extracts were parallel to the standard curve. The intra-assay and inter-assay coefficients of variation were 7% and 9% respectively.
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Subcellular Fractionafion Gastric fundic mucosa was incubated at 37~ for 30min and then subsequently washed twice in order to remove endogenous somatostatin. Cytosol of gastric fundic mucosa was isolated according to Reyl-Desmars and Lewin (6), cyr.osol being considered as the supernatant obtained at 105,000g. Protein was estimated by the method of Lowry et al. (16), using bovine serum albumin as a standard. Binding Studies Binding studies were performed in optimal conditions according to a previously reported technique (6). Briefly, a cytosolic fraction of rabbit gastric fundic mucosa (0.2 mg protein/ml) was incubated at 25~ for 60 min in 0.5 ml of medium (pH 7.4) of the following composition: 0.5mMNaH2PO4, 1 raM NA2HPO4, 80 mM NaC1, 5,mM KC1, 1 mM CaCI2, 1.5 mM MgCl2, 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 11 mM glucose, 0.1% bovine serum albumin, 0.1 mg/ml trypsin inhibitor and 50pM 125I-Tyrll somatostatin either alone or together with increasing concentrations of unlabelled somatostatin (up to 2/zM). Unbound peptide was removed by adding 1 ml of 0.25% activated charcoal, 0.5% bovine serum albumin and 0.025% T-70 dextran (6). "Specific" binding was estimated as the difference between "total" binding (i.e. in the presence of tracer alone) and "non-specific" binding (as measured in the presence of 4 #M unlabelled somatostatin). This non-specific component represented about 45% of the binding observed in the absence of unlabelled somatostatin. The integrity of bound lzSI-Tyr1~-somatostatin was observed by talc absorption (17). Statistial Analysis Comparison between groups was performed by Student's t test for unpaired data. Statistical significance was considered when p < 0.05.
RESULTS Development of the Diabetic State Rabbits receiving alloxan (n =5) underwent a progressive weight loss (2--3 Kg at the time of drug injection and 1.2-1.8 Kg three months later). Two days after alloxan administration, the 24 h urine volumes in the diabetic rabbits were significantly greater than in the control group (n =5). Polyuria and glucosuria were maintained thereafter until sacrifice of the rabbits three months after commencement of the study. The fasting blood glucose at the time of sacrifice was significantly higher in the diabetic (458 + 34 mg/100 ml) than in the
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control group (78+8mg/100ml). Plasma insulin was significantly lower in diabetic rabbits (5.5+0.9/tU/ml) as compared to control animals (37.7+ 4.7/~U/ml). Concentration of SLI in Gastric Fundic Mucosa and Plasma The SLI content in gastric fundic mucosa was significantly higher in diabetic rabbits than that in control animals (Fig. 1). As shown in Fig. 1, fasting plasma SLI levels were higher in the portal vein than in the aorta (p < 0.005) and aortic levels were greater than those in the inferior vena cava (p
