Upsala J Med Sci 97: 157-167 Effects of Omeprazole and Ranitidine on Plasma Gastrin Concentration and Stomach Gastrin Content in Rats Rein Seensalu,' Kinfe Girma,2 Bengt Romel12 and Goran Nilsson2 'Division of Gastroenterology & Hepatology, Department of Medicine and Department of Surgery and Research Center, Karolinska institute, Huddinge University Hospital, Huddinge and 2Department of Physiology, Faculty of Veterinary Medicine, University of Agricultural Sciences, Uppsala, Sweden ABSTRACT Prolonged fasting and longer time between dosing and sampling reduced the plasma gastrin concentrations after omeprazole (80 pmol/kg x 2 for 14 days) treatment in male rats whereas the amounts of tissue gastrin were essentially unchanged during these initial experiments. After 28 days omeprazole (80 pmol/kg x 2) or ranitidine (375 pmol/kg x 4) that produced corresponding inhibition of acid secretion, increased the tissue gastrin content by 114 and 59 %. A low dose of omeprazole (20 pmol/kg x 2) also raised the gastric gastrin content (41 %), whereas no change was noted on treatment with a low dose of ranitidine (125 pmol x 4). Following recovery for 28 days no significant increases in gastrin were observed. 1, 3, 7, 14 or 28 days of treatment with omeprazole (80 pmol/kg x 2) gradually increased the gastric gastrin content beeing significantly raised already after 3 days. We conclude that a) measuring the tissue gastrin content may be the preferable method when changes in gastrin following long-term treatment with acid inhibiting drugs are to be determined, b) the amount of gastrin in the stomach increases rapidly following treatment with omeprazole and is approximately doubled following 28 days of treatment and c) after treatment for 28 days omeprazole was found to cause greater elevations in the tissue gastrin content than ranitidine despite similar degrees of basal acid inhibition. INTRODUCTION It is well known from animal and human studies that a reduction of the acid output will increase the plasma gastrin concentration (3, 6, 12, 14,23). The elevated secretion of gasmn will exert a trophic effect on the acid-producing portion of the stomach (8, 14) and may also increase the number of fundic ECL (enterochromaffin-like) cells (10-1 1, 20-22, 27). In addition, long-term treatment with some acid-suppressing drugs has produced carcinoid (4, 5, 7, 19) or other (26) gastric tumours in rats. The Occurrence of carcinoid tumours in rats following treatment with the selective H+/K+-ATPase inhibitor omeprazole has been ascribed to the elevated intragasmc pH and the subsequent increase in gastrin release (4). However, the concept that an increased antral 157 pH during treatment with acid suppressing drugs is the only factor responsible for the gastrin increase is questioned by others (3, 16-18,25). Thus, further studies are motivated on such drugs, on gastrin production and release. In the present investigation we have therefore studied the influence of the H2-receptor antagonist ranitidine and omeprazole on tissue and plasma concentrations of gastrin, using doses of ranitidine and omeprazole that evoke similar degrees of inhibition of the basal acid output. Acid secretory experiments were performed in this study to confirm the inhibitory effects on acid secretion found in a previous investigation by us (24). In the present study we also investigated how low and high doses of omeprazole and ranitidine affect the tissue gastrin concentration following four weeks of treatment and four weeks of recovery from such treatment. We also determined how the gastric tissue concentration gradually changes during the treatment period with a high dose of omeprazole. The effects of H2-receptor antagonists (5, 10,20-23,27, ) and omeprazole (2, 3, 8,9-10,20-23, 27) on gastrin concentrations in rats have been studied previously. In many of those studies gastrin concentrations in plasma and tissues were determined under different conditions, which complicates comparisons between the studies. To evaluate factors that are of importance in this context we have studied the influence on plasma and tissue gastrin concentrations during omeprazole treatment when the animals have free access to food and during different periods of food deprivation, as well as the effect of changing the interval between the last dose and sample collection. MATERIALS AND METHODS Animals Male Sprague-Dawley rats, weighing 200-325 g were used in the acid secretory experiments. The rats were provided with a stainless steel cannula in the proximal part of the stomach under pentobarbitone anaesthesia. After surgery the rats were allowed to recover for three weeks before acid secretory experiments were started. Before each experiment the rats had free access to water but were fasting for 18-20 hours. Male Sprague-Dawley rats weighing 170-200 g at the start of the experiments were used in the gastrin studies. The animals were weighed twice a week and the average weight gain was calculated. The rats, all of which had free access to water, were fed with standard rat food (R3, Ewos AB, Sijdertalje, Sweden) containing 22.0 % protein, 51.5 % carbonhydrate and 5.0 % fat, 13 MJ/kg. When fasting, rats had free access to water and were placed in mesh wire bottom cages 2 - 24 hours before they were killed. 158 Drugs and drug administration The drugs omeprazole ( generously supplied by AB Hassle, Sweden) and ranitidine (Glaxo Operations UK Ltd., England) were dispersed or dissolved in hydroxypropylmethylcellulose (Dow Coming Corp., Midland, USA). When hydroxypropylmethylcellulose (Methocel) was used as vehicel or in control experiments 2 m d m l of NaHCO3 was dissolved in 0.5% of Methocel and NaOH added in order to adjust pH to 9.0. The omeprazole suspensions were kept in a refrigerator (+4OC) and renewed every 6th day. Fresh ranitidine solutions were prepared twice weekly to assure stability. On the basis of body weight determinations, the amounts of drugs to be administered were regularly adjusted. Drugs were given orally by gavage with a flexible plastic tube in volumes varying between 2.7 and 3.3 ml. In all experiments the drugs or the vehicle were always administered twice (omeprazole at 7 am and 7 pm) or four (ranitidine at 1 am, 7 am, 1 pm and 7 pm) times daily in order to accomplish a persistent 24 hour inhibition during the treatment period. Samples from 9-10 rats were collected for each observation on gastrin concentrations. Treatment of plasma and gastric tissue After conclusion of the drug administration, rats were killed by decapitation. Blood was collected from the jugular and carotid vessels in heparinized glass tubes, cooled and centrifuged (5000 rpm) for 10 minutes at +4OC. The plasma was then frozen and stored at -20% After bleeding, the abdominal wall was opened by a midline incision and the stomach was taken out. Blood vessels and mesenteric tissue along the major and minor curvatures were removed. The stomach was opened along the major curvature and gently rinsed with saline. Following determination of the weight the stomach was frozen on dry ice and then stored at -2OOC until assayed for gastrin content by radioimmunoassay. The gasmn content of the whole stomach was determined. The stomach was homogenized and then boiled in water for 10 minutes (w/v=l/lO). After cooling, the mixture was filtered through gauze and the filtrate was adjusted to 25 ml and then stored at -2OOC. Radioimmunological procedures Gastrin was determined by radioimmunoassay according to a method described previously (13, using antibodies (4562) generously supplied by Professor Jens Rehfeld, Copenhagen, Denmark. Synthetic human gastrin 17 I (generously supplied by Dr J S Morley, ICI Ltd., England) was used as standard. Human gastrin I labelled with 1125, purchased from Milab, Malmo, Sweden, was used as tracer. Gasmn determinations were performed in duplicate and in serial dilutions. Plasma gastrin concentrations are expressed in p d m l and the gastrin amounts in tissues in pgstomach. 159 Acid secretion studies Before acid secretory experiments the gastric cannula was opened and carefully rinsed to allow a free passage of gastric juice. Basal and pentagastrin (650 nmol/kg/h in S.C. infusion) stimulated gastric acid secretions were followed for 24 hours in rats given ranitidine (375 pmol/kg every sixth hour), omeprazole (80 pmol/kg every twelfth hour) or vehicle (3.0 ml, 2 or 4 times daily). On the seventh day, during ongoing drug administration, the acid secretion was determined in ranitidine treated rats hours 3,6,9,12,15,18,21 and 24 or during hours 4,8,12,16,20 and 24 in rats given omeprazole. Pentagastrin was given continously during the hours of sample collection. At collection of gastric juice the volume was measured and the acidity was determined by titration with 0.1 M NaOH to pH 7.0 using an automatic endpoint titrator system (Radiometer, Copenhagen, Denmark). Gastrin studies Before starting other studies, we determined how gastrin in plasma and stomach tissue were influenced by the conditions under which samples were collected. Thus we examined how feeding and food deprivation for various periods of time influenced the concentrations (series A). We also studied how variations in the interval between the last drug administration and sampling affected the gastrin concentrations (series B). The results from these initial studies were used in planning for more extensive studies in which the stomach gastrin content was determined after 4 weeks of treatment with omeprazole and ranitidine and following a subsequent recovery period of 4 weeks (series C). In series D we investigated how a high dose of omeprazole influences gastric gastrin content following 1,3,7, 14 and 28 days of administration. Statistical evaluations All values are expressed as mean k SEM. Statistical evaluation of the results in series A and B was carried out by regression analysis. In series C analysis of variance and Bonferroni's test was carried out for multipel comparisons and in series D Student's t-test was used for comparisons between groups. RESULTS Ranitidine (375 pmolkg x 4) inhibited the 24 hour basal and pentagastrin stimulated acid secretions with 82k2% (mean+SEM) and 7 9 f l % (fig. la). The corresponding inhibition for omeprazole (80 pmolkg x 2) was 8 M 5 % and 78k2% (fig. 1 b). 160 Fig. 1. Basal (A A ) and pentagastrin stimulated ( 0 0) acid out-put during 24 hours in gastric fistula rats (n 6-10) during the seventh day of administration of a) 375 pmol/kg (A@) ranitidine or 3.0 ml of Methowl( A 0 ) at hours 0,6, 12 and 24 or during administration of b) 80 pmol/kg of omeprazole (A 0 ) or 3.0 ml of Methocel (AO) at hours 0 and 12. Gastric juice were collected at hours 3,6,9,12,15,18,2 1,24 (ranitidine) or 4,8,12,16,20, 24 (omeprazole). 400 1 - 2oo l-LAkf& 4 8 1 2 16 20 2 4 HOURS No significant differences in body or stomach weights were seen between the different treatment groups in series A - C (results not illustrated). In series A rats were given omeprazole (80 pmoVkg x 2) for 14 days and were killed 12 hours after the last dose of omeprazole. The animals had access to food until killed (0) or they had been fasting for 2, 6, 12 or 24 hours. As can be seen in Fig. 2a, plasma gastrin concentrations gradually and significantly decreased as the period of fasting was prolonged (t=-7.29, p10.001), whereas the amounts in gastric tissue (Fig. 2b) at the corresponding points of time were essentially unchanged (t=1.65, NS). The rats in series B had free access to food and the concentration of gastrin in plasma was significantly (t=-5.29, p10.001) lowered as time between dosing and sampling was prolonged (Fig. 3a). The amounts of gasmn in the stomach were not greatly influenced (t=1.75, NS), although some increase of gastrin content was seen as the period between sampling and last administration of the drug was increased (Fig. 3b). The time point 0 hour after drug administration was omitted in the statistical analysis. The rationale for that is that the time between administration and sampling of plasma and stomach tissue was too short to allow omeprazole to interfere with acid and gastrin secretion. Effects of treatment with omeprazole (20 or 80 pmol/kg x 2) or ranitidine (125 or 375 pmolkg x 4) for 28 days and following 28 days of recovery were studied in series C . After 18-20 hours of fasting the treated animals were killed 6 (ranitidine) or 12 (omeprazole) hours after the last dose. The high dose of omeprazole (Fig. 4) and ranitidine (Fig. 5 ) significantly increased the tissue content of gastrin. The largest amounts were found when animals were given the high omeprazole dose for four weeks. However, the high dose of ranitidine caused a gastrin increase that was closer to that produced by the low dose of omeprazole. 161 No change in tissue gastrin content was noted in rats treated with the low dose of ranitidine (Fig. 5). After 4 weeks of recovery, the amounts of tissue gastrin in omeprazole treated rats were, somewhat, but not significantly, raised whereas the amounts in ranitidine-treated rats were unchanged when compared to the recovery controls. T 0 2 6 12 24 HOUrS 0 2 6 1 2 2 4 H O U E 0 2 6 1 2 2 4 Hours 0 2 6 1 2 2 4 Hours Fig 2. (a) Plasma gastrin concentration and (b) gasmc gastrin content, expressed as mean k SEM, in rats treated with omeprazole 80 pmolkg twice daily for 14 days. The rats (n=9-10) were killed after having free access to food (0 hour) or after 2, 6, 12 or 24 hours of food deprivation. Fig 3. (a) Plasma gastrin concentration and (b) gastric gasmn content in rats having free access to food (n=9-10) treated with omeprazole 80 prnolkg twice daily for 14 days and killed 0,2,6,12 or 24 hours after the administration of the last dose. Results are expressed as mean If: SEM. 162 Treatment lor 28 days Recovery for 28 days ... ,1 T Treatment for 28 days Recovery for 28 days 3 1 ... - P ... - Methocel I Omeorazole I 1 3 7 Days of treatment 1 4 Fig 4. Stomach gastrin content in rats (n=10) treated with 20 or 80 pmol/kg of omeprazole or with 3 ml of Methocel every twelfth hour for 28 days and following recovery for 28 d a y s . T h e r e s u l t s a r e expressed as mean k SEM. *** indicates significant ( ~ 1 0 . 0 0 1 ) difference between Methocel and omeprazole- treated rats. Bonferroni’s test. Fig 5. Stomach gastrin content in rats (n=10) treated with 125 or 375 p m o l k g of ranitidine or with 3 ml of Methocel every sixth hour for 28 days and following recovery for 28 days. *** indicates significant (p