%,urosciem'e Lvtler~', 145 (1992) 229 233 c, 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
229
NSL 09020
Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum M i n e k o F u j i m i y a ~', C h r i s t o p h e r H.S. M c l n t o s h ~, H i r o s h i K i m u r a b a n d Yin N a m K w o k ~ "D~7~artment ol 4natomy, I'lnstitute o['Molecular Neurohiolog3', Shiga University o/Medical Science, Shiga (Japan) and'MR(" Reguhttory Pepti~h" Group, Department ()/'Physiology, [,)uver~'ily o1' British ('olumhia, l'ancouver, B. C. (('amuht) (Received 25 November 1991: Revised version received 20 July 1992: Accepted 20 July 1992)
Key word~v Rat duodenum: Somatostatin-like immunoreactivity; Luminal release: Somatostatin-28: Somatostatin-14: Carbachol The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumcn was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SL1 from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about I during basal release but increased to approximately 2 during carbachol stimulation.
Somatostatin in the gastrointestinal (GI) tract is well known to be present in epithelial D cells as well as in nerve fibers of the enteric plexus and to play an inhibitory role in the control of motility, exocrine and endocrine secretions [2]. However, it is unclear whether the somatostatin exerts a paracrine or direct action on the target cells, or acts through an endocrine or neurohormonal mechanism. In previous studies on release of somatostalin-like immunoreactivity (SLI) from the GI tract, the hormonal release into the vascular system has been extensively examined, and the experimental models of the isolated, vascularly perfused stomach [11, 12] or intestine [8] have been preferentially used. Although there has been no dependable method for studying potential paracrine effects of the peptide, SLI release into the antral lumen of rat [1,9] or human [6] has been demonstrated, with the released SLI being thought to exert its action in a paracrine fashion. Recently, the release of SLI into the lumen from rat duodenum has been also demonstrated [17]. In these previous studies, however, the only pharmacological experiments on the luminal SLI release were performed using systemic administration of the drugs. The present study aimed at examining the possible control by cholinergic mechanisms on the luminal release of SLI in rat duodenum, since this mechCorre.q~ondence." M. Fujimiya, Department of Anatomy, Shiga University of Medical Science, Seta, Otsu. Shiga, 510-21, Japan. Fax: (81) (775) 43-4995.
anism has been known to give predominantly an excitatory control on GI functions [5]. We used the isolated, vascularly perfused duodenum because it allowed administration of a known concentration of a cholinergic agonist or antagonist directly into the duodenal segment through the vascular perfusate. Thirty male Wistar rats, weighing 250 350 g were used. The animals were housed in a light-controlled room with generally free access to laboratory food and water, but were fasted overnight before the operation. The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The duodenum was prepared for both vascular and intraluminal perfusion. The procedure used to prepare t\)r duodenal perfusion was similar to that for gastric perfusion [10 12], with the following slight modifications. Vascular perfusion was achieved through an aortic cannula with the tip lying adjacent to the superior mesenteric arteries, and effluent was collected through a portal vein cannula. All vasculature apart from that leading into the duodenum were cut between double ligatures. The stomach, small intestine, colon, pancreas and spleen were carefully separated from the duodenum and removed. Luminal perfusion was performed through a cannula inserted into the pylorus; effluent perfusate was collected through a cannula placed into the duodenal lumen at the level of Treitz" ligament. The vascular perfusate consisted of a mixture of Krebs' solution, 3% dextran and 0.2% bovine serum al-
23O
bumin. The perfusate was saturated with 95% O~/5% CO2 gas to maintain a pH of 7.4. A saline solution was used as a luminal perfusate. Both perfusates and the preparation were kept at 37°C throughout the experiment by thermostatically controlled heating apparatus. The flow rates for vascular and luminal perfusion were maintained 3 ml/min and 1 ml/min, respectively. After a 25-rain equilibration period, luminal effluents were collected at 5-min intervals into ice-cold vials containing 200 kIU/ml of aprotinin (Trasylol; Miles) to prevent the degradation of SLI [16]. Carbachol (carbamylcholine chloride; Aldrich), atropine sulfate (Sigma) and hexamethonium bromide (Sigma) were introduced into the vasculature via a side arm infusion at final concentrations as indicated in the text. Each specimen from the collecting vials was applied to a Sep-Pak Ct~ cartridge (Waters). The fractionation method was as previously described [t0], except that SLI was eluted by 2 ml of 60% acetonitrile containing 0.1% trifluoroacetic acid (TFA). Under the conditions used over 85% of SLI was recovered. The eluate was lyophilized and then reconstituted with an assay buffer solution for measuring SLI [14] using a monoclonal antibody (SOMA 03). As previously shown, this antibody has equipotent detection for both somatostatin-14 and -28. Results are expressed as mean _+ S.E.M. in pg/min of samples collected for 5 min. In the experiment using carbachol infusion, the mean basal release (average of values before carbachol infusion) and mean SLI release induced by carbachol (average of values within a period of carbachol infusion) were calculated. The effect of carbachol was expressed as a % stimulation which was calculated as 100 x (mean SLI release induced by carbachol-mean basal release)/mean basal release. High performance liquid chromatography (HPLC) analysis was done on perfusates obtained from 3 experiments on the stimulation of SLI release by 1 pM carbachol. Samples collected from 0-15 min (before carbachol infusion), 15-35 min (with carbachol infusion) and 35-50 min (carbachol withdrawal periods) were analyzed separately. For HPLC, a p-Bondapak Ct8 column was used, and somatostatin-14 and somatostatin-28 were clearly separated by using a 26-33% gradient of acetonitrile in water containing 0.1% TFA at a flow rate of 1 ml/min [13]. Detection was performed at 214 nm. The HPLC eluate was collected every 0.5 min, lyophilized and dissolved in assay buffer for SLI radioimmunoassay. Synthetic somatostatin-14 and -28 were separately applied to the column and their elution positions were determined. Statistical analysis of the data described in Fig. 1 was performed using a single factor ANOVA for repeated measures followed by the Dunnett t-test. A paired t-test
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Fig. 1. Luminal SLI release either with carbachol alone (o, n--4) or with atropine + carbachol (©, n=5). Each value represents mean _+ S,E.M. of samples collected over 5 min. x axis shows the time measured from the start of sample collection. Carbachol (1 # M ) was infused either from 15 to 35 min (when used alone) or from 20 to 35 min (when used with atropine). Atropine (1 p M ) was infused from 15 to 35 min. The SLI release levels marked with asterisks were significantly (P < 0,05) increased as compared with the value marked with an a.
was used to compare the values of mean basal release and mean SLI release induced by carbachol in Table I. In both cases, a P value of
229
NSL 09020
Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum M i n e k o F u j i m i y a ~', C h r i s t o p h e r H.S. M c l n t o s h ~, H i r o s h i K i m u r a b a n d Yin N a m K w o k ~ "D~7~artment ol 4natomy, I'lnstitute o['Molecular Neurohiolog3', Shiga University o/Medical Science, Shiga (Japan) and'MR(" Reguhttory Pepti~h" Group, Department ()/'Physiology, [,)uver~'ily o1' British ('olumhia, l'ancouver, B. C. (('amuht) (Received 25 November 1991: Revised version received 20 July 1992: Accepted 20 July 1992)
Key word~v Rat duodenum: Somatostatin-like immunoreactivity; Luminal release: Somatostatin-28: Somatostatin-14: Carbachol The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumcn was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SL1 from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about I during basal release but increased to approximately 2 during carbachol stimulation.
Somatostatin in the gastrointestinal (GI) tract is well known to be present in epithelial D cells as well as in nerve fibers of the enteric plexus and to play an inhibitory role in the control of motility, exocrine and endocrine secretions [2]. However, it is unclear whether the somatostatin exerts a paracrine or direct action on the target cells, or acts through an endocrine or neurohormonal mechanism. In previous studies on release of somatostalin-like immunoreactivity (SLI) from the GI tract, the hormonal release into the vascular system has been extensively examined, and the experimental models of the isolated, vascularly perfused stomach [11, 12] or intestine [8] have been preferentially used. Although there has been no dependable method for studying potential paracrine effects of the peptide, SLI release into the antral lumen of rat [1,9] or human [6] has been demonstrated, with the released SLI being thought to exert its action in a paracrine fashion. Recently, the release of SLI into the lumen from rat duodenum has been also demonstrated [17]. In these previous studies, however, the only pharmacological experiments on the luminal SLI release were performed using systemic administration of the drugs. The present study aimed at examining the possible control by cholinergic mechanisms on the luminal release of SLI in rat duodenum, since this mechCorre.q~ondence." M. Fujimiya, Department of Anatomy, Shiga University of Medical Science, Seta, Otsu. Shiga, 510-21, Japan. Fax: (81) (775) 43-4995.
anism has been known to give predominantly an excitatory control on GI functions [5]. We used the isolated, vascularly perfused duodenum because it allowed administration of a known concentration of a cholinergic agonist or antagonist directly into the duodenal segment through the vascular perfusate. Thirty male Wistar rats, weighing 250 350 g were used. The animals were housed in a light-controlled room with generally free access to laboratory food and water, but were fasted overnight before the operation. The animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The duodenum was prepared for both vascular and intraluminal perfusion. The procedure used to prepare t\)r duodenal perfusion was similar to that for gastric perfusion [10 12], with the following slight modifications. Vascular perfusion was achieved through an aortic cannula with the tip lying adjacent to the superior mesenteric arteries, and effluent was collected through a portal vein cannula. All vasculature apart from that leading into the duodenum were cut between double ligatures. The stomach, small intestine, colon, pancreas and spleen were carefully separated from the duodenum and removed. Luminal perfusion was performed through a cannula inserted into the pylorus; effluent perfusate was collected through a cannula placed into the duodenal lumen at the level of Treitz" ligament. The vascular perfusate consisted of a mixture of Krebs' solution, 3% dextran and 0.2% bovine serum al-
23O
bumin. The perfusate was saturated with 95% O~/5% CO2 gas to maintain a pH of 7.4. A saline solution was used as a luminal perfusate. Both perfusates and the preparation were kept at 37°C throughout the experiment by thermostatically controlled heating apparatus. The flow rates for vascular and luminal perfusion were maintained 3 ml/min and 1 ml/min, respectively. After a 25-rain equilibration period, luminal effluents were collected at 5-min intervals into ice-cold vials containing 200 kIU/ml of aprotinin (Trasylol; Miles) to prevent the degradation of SLI [16]. Carbachol (carbamylcholine chloride; Aldrich), atropine sulfate (Sigma) and hexamethonium bromide (Sigma) were introduced into the vasculature via a side arm infusion at final concentrations as indicated in the text. Each specimen from the collecting vials was applied to a Sep-Pak Ct~ cartridge (Waters). The fractionation method was as previously described [t0], except that SLI was eluted by 2 ml of 60% acetonitrile containing 0.1% trifluoroacetic acid (TFA). Under the conditions used over 85% of SLI was recovered. The eluate was lyophilized and then reconstituted with an assay buffer solution for measuring SLI [14] using a monoclonal antibody (SOMA 03). As previously shown, this antibody has equipotent detection for both somatostatin-14 and -28. Results are expressed as mean _+ S.E.M. in pg/min of samples collected for 5 min. In the experiment using carbachol infusion, the mean basal release (average of values before carbachol infusion) and mean SLI release induced by carbachol (average of values within a period of carbachol infusion) were calculated. The effect of carbachol was expressed as a % stimulation which was calculated as 100 x (mean SLI release induced by carbachol-mean basal release)/mean basal release. High performance liquid chromatography (HPLC) analysis was done on perfusates obtained from 3 experiments on the stimulation of SLI release by 1 pM carbachol. Samples collected from 0-15 min (before carbachol infusion), 15-35 min (with carbachol infusion) and 35-50 min (carbachol withdrawal periods) were analyzed separately. For HPLC, a p-Bondapak Ct8 column was used, and somatostatin-14 and somatostatin-28 were clearly separated by using a 26-33% gradient of acetonitrile in water containing 0.1% TFA at a flow rate of 1 ml/min [13]. Detection was performed at 214 nm. The HPLC eluate was collected every 0.5 min, lyophilized and dissolved in assay buffer for SLI radioimmunoassay. Synthetic somatostatin-14 and -28 were separately applied to the column and their elution positions were determined. Statistical analysis of the data described in Fig. 1 was performed using a single factor ANOVA for repeated measures followed by the Dunnett t-test. A paired t-test
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Fig. 1. Luminal SLI release either with carbachol alone (o, n--4) or with atropine + carbachol (©, n=5). Each value represents mean _+ S,E.M. of samples collected over 5 min. x axis shows the time measured from the start of sample collection. Carbachol (1 # M ) was infused either from 15 to 35 min (when used alone) or from 20 to 35 min (when used with atropine). Atropine (1 p M ) was infused from 15 to 35 min. The SLI release levels marked with asterisks were significantly (P < 0,05) increased as compared with the value marked with an a.
was used to compare the values of mean basal release and mean SLI release induced by carbachol in Table I. In both cases, a P value of
