Pharmacological Research, Vol. 22, No. 5, 1990
605
TACHYKININS: EFFECTS ON GASTRIC SECRETION AND EMPTYING IN RATS GIOVANNA IMPROTA and MARIA BROCCARDO
Institute of Pharmacology Ill, University 'La Sapienza', Rome, Italy Received in final form 29 January 1990
SUMMARY To test the effects on gastric acid secretion and gastric emptying of non-mammalian (eledoisin, physalaemin, kassinin), mammalian (substance P, neurokinin A, neurokinin B) and synthetic tachykinins (septide, senktide), intracerebroventricular injections of these peptides were given to conscious pylorus-ligated rats (gastric secretory study) or to animals fed with a phenol red meal (gastric emptying study). The tachykinins able to cause central inhibition of acid output were those active on NK2 and NK3 receptors (eledoisin, kassinin, neurokinin A, neurokinin B and senktide). Tachykinins active on NK1 receptors (substance P, physalaemin and septide) were devoid of this activity. The most potent inhibitor was the synthetic NK3 agonist, senktide. All tested natural tachykinins significantly inhibited gastric emptying, the most potent being those active on NK2 receptors. Septide, the synthetic NK1 agonist, had lowest effect and senktide, the synthetic NK3 agonist, was far less active than eledoisin, kassinin and neurokinin A. Thus, specific tachykinin receptors exist in the rat brain that mediate gastric functions: NK3 receptors participate in the control of gastric secretion, while NK2 receptors seem to be involved in the regulation of gastric emptying by activating inhibitory pathways.
KEYWORDS:natural and synthetic tachykinins, CNS control, acid output, gastric emptying.
INTRODUCTION
Tachykinins (TKs) represent a large family of peptides with the common carboxylterminal amino acid sequence Phe-X-Gly-Leu-Met-NH2 and with widespread distribution in mammalian and non-mammalian tissues [1, 2]. TKs share a similar spectrum of biological activities, but can be subdivided on quantitative terms. The occurrence of differing rank orders of TK potencies on smooth muscle preparations and ligand binding assays, demonstrates the existence both in peripheral organs and in the CNS of multiple TK receptors: one for substance P (SP), NK1 receptor, one for neurokinin A (NKA), NK2 receptor, and one for neurokinin B (NKB), NK3 receptor [3-5]. The non-mammalian TKs also bind potently to TK receptors in mammalian CNS: eledoisin (EL) and kassinin (KS) are 1043-6618/90/050605-06/S03.00/0
© 1990 The Italian Pharmacological Society
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Pharmacological Research, Vol. 22, No. 5, 1990
more active on NK2 and NK3 subtypes; physalaemin (PH) is active on the NK1 subtype [6]. In addition to the natural TKs, two synthetic TK analogues have recently been reported to possess high selectivity for TK-receptor subtypes: septide (SPT) interacts with the NK1 receptor and senktide (SNK) with the NK3 receptor [7]. Because TK receptors are distinct entities, which mediate different functions, the natural and synthetic selective agonists are important tools for studies on the role of the TK-ergic system. Though functional evidence for the three receptors has been provided only for the peripheral effects of TKs, recent reports on drinking behaviour and salt appetite suggest the involvement of three central receptor subtypes similar to those in the periphery [8]. Because SP may also modulate gastrointestinal functions [9-13] and, to our knowledge, no systematic study has determined the role of TKs on the CNS control of gastri c secretion and emptying, the aim of the present study was to evaluate gastric secretory and motor responses to i.c.v, injections of different TKs and to identify in the rat CNS TK-receptor subtypes that play a role in the control of gastric acid secretion and motility.
MATERIAL AND METHODS
Male Wistar rats weighing 200-250 g, to be injected intracerebroventricularly (i.c.v.), were implanted with a permanent Plexiglass cannula in the left lateral ventricle, 7 days before they were studied. After surgery, they were caged individually on wire mesh to prevent coprophagia and were fasted, but had free access to water for 48 h before the experiment. The animals were separated into two groups according to the experimental study performed. Gastric secretion
At the time of the experiment, the animals were lightly anaesthetized with diethyl ether, the abdomen was opened and the pylorus ligated; care was taken not to interfere with the blood supply to the stomach. The abdomen was then closed with two interrupted sutures; 30 min later, when the rats had completely regained the righting reflex, they were i.c.v, injected with different doses of the peptides in a constant volume of 5 ktl, or with saline. The following substances and doses (nmol/ rat) were used: EL, 0.06, 0.6 and 6; KS, 0,07, 0.7 and 7; PH, 0.7, 7 and 35; SP, 0.06, 0.6 and 6; NKA, 8, 16 and 40; NKB, 0.35, 3.5 and 7; SPT, 10, 20 and 50; SNK, 0.0004, 0.004, 0.008 and 0.08. The rats were decapitated 120 min after the peptide injection; the abdomen was opened, the stomach removed, washed with 5 ml of distilled water and the contents were collected and centrifuged. The volume of gastric content was recorded and the hydrogen concentration determined by titration to pH 7.0 with 0.01 M NaOH using a Crison microTT 2022 automatic titrator. Gastric acid output was expressed as per cent of the output in control rats.
Pharmacological Research, Vol. 22, No. 5, 1990
607
Gastric emptying In the gastric emptying assay, we used a phenol red meal given by oral intubation; the peptides were given at meal time and 60 min later the rats were then sacrificed. The stomach was removed and submitted to the phenol red assay described elsewhere [14]. The results are presented as per cent changes with respect to control rats. The following substances and doses (nmol/rat) were used: EL, 0.06, 0.6 and 6; KS, 0.007, 0.07, 0.7 and 7; PH, 0.6, 6 and 9; SP, 0.06, 0.6 and 6; NKA, 0.08, 0.8 and 8; NKB, 0.06, 0.6 and 3.5; SPT, 1.2 and 11.2; SNK, 0.9, 6 and 9. Data were analysed for statistical significance on an Apple II computer using ANOVA and the Duncan test for post hoc data analysis.
120 SPT
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ao
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60
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605
TACHYKININS: EFFECTS ON GASTRIC SECRETION AND EMPTYING IN RATS GIOVANNA IMPROTA and MARIA BROCCARDO
Institute of Pharmacology Ill, University 'La Sapienza', Rome, Italy Received in final form 29 January 1990
SUMMARY To test the effects on gastric acid secretion and gastric emptying of non-mammalian (eledoisin, physalaemin, kassinin), mammalian (substance P, neurokinin A, neurokinin B) and synthetic tachykinins (septide, senktide), intracerebroventricular injections of these peptides were given to conscious pylorus-ligated rats (gastric secretory study) or to animals fed with a phenol red meal (gastric emptying study). The tachykinins able to cause central inhibition of acid output were those active on NK2 and NK3 receptors (eledoisin, kassinin, neurokinin A, neurokinin B and senktide). Tachykinins active on NK1 receptors (substance P, physalaemin and septide) were devoid of this activity. The most potent inhibitor was the synthetic NK3 agonist, senktide. All tested natural tachykinins significantly inhibited gastric emptying, the most potent being those active on NK2 receptors. Septide, the synthetic NK1 agonist, had lowest effect and senktide, the synthetic NK3 agonist, was far less active than eledoisin, kassinin and neurokinin A. Thus, specific tachykinin receptors exist in the rat brain that mediate gastric functions: NK3 receptors participate in the control of gastric secretion, while NK2 receptors seem to be involved in the regulation of gastric emptying by activating inhibitory pathways.
KEYWORDS:natural and synthetic tachykinins, CNS control, acid output, gastric emptying.
INTRODUCTION
Tachykinins (TKs) represent a large family of peptides with the common carboxylterminal amino acid sequence Phe-X-Gly-Leu-Met-NH2 and with widespread distribution in mammalian and non-mammalian tissues [1, 2]. TKs share a similar spectrum of biological activities, but can be subdivided on quantitative terms. The occurrence of differing rank orders of TK potencies on smooth muscle preparations and ligand binding assays, demonstrates the existence both in peripheral organs and in the CNS of multiple TK receptors: one for substance P (SP), NK1 receptor, one for neurokinin A (NKA), NK2 receptor, and one for neurokinin B (NKB), NK3 receptor [3-5]. The non-mammalian TKs also bind potently to TK receptors in mammalian CNS: eledoisin (EL) and kassinin (KS) are 1043-6618/90/050605-06/S03.00/0
© 1990 The Italian Pharmacological Society
606
Pharmacological Research, Vol. 22, No. 5, 1990
more active on NK2 and NK3 subtypes; physalaemin (PH) is active on the NK1 subtype [6]. In addition to the natural TKs, two synthetic TK analogues have recently been reported to possess high selectivity for TK-receptor subtypes: septide (SPT) interacts with the NK1 receptor and senktide (SNK) with the NK3 receptor [7]. Because TK receptors are distinct entities, which mediate different functions, the natural and synthetic selective agonists are important tools for studies on the role of the TK-ergic system. Though functional evidence for the three receptors has been provided only for the peripheral effects of TKs, recent reports on drinking behaviour and salt appetite suggest the involvement of three central receptor subtypes similar to those in the periphery [8]. Because SP may also modulate gastrointestinal functions [9-13] and, to our knowledge, no systematic study has determined the role of TKs on the CNS control of gastri c secretion and emptying, the aim of the present study was to evaluate gastric secretory and motor responses to i.c.v, injections of different TKs and to identify in the rat CNS TK-receptor subtypes that play a role in the control of gastric acid secretion and motility.
MATERIAL AND METHODS
Male Wistar rats weighing 200-250 g, to be injected intracerebroventricularly (i.c.v.), were implanted with a permanent Plexiglass cannula in the left lateral ventricle, 7 days before they were studied. After surgery, they were caged individually on wire mesh to prevent coprophagia and were fasted, but had free access to water for 48 h before the experiment. The animals were separated into two groups according to the experimental study performed. Gastric secretion
At the time of the experiment, the animals were lightly anaesthetized with diethyl ether, the abdomen was opened and the pylorus ligated; care was taken not to interfere with the blood supply to the stomach. The abdomen was then closed with two interrupted sutures; 30 min later, when the rats had completely regained the righting reflex, they were i.c.v, injected with different doses of the peptides in a constant volume of 5 ktl, or with saline. The following substances and doses (nmol/ rat) were used: EL, 0.06, 0.6 and 6; KS, 0,07, 0.7 and 7; PH, 0.7, 7 and 35; SP, 0.06, 0.6 and 6; NKA, 8, 16 and 40; NKB, 0.35, 3.5 and 7; SPT, 10, 20 and 50; SNK, 0.0004, 0.004, 0.008 and 0.08. The rats were decapitated 120 min after the peptide injection; the abdomen was opened, the stomach removed, washed with 5 ml of distilled water and the contents were collected and centrifuged. The volume of gastric content was recorded and the hydrogen concentration determined by titration to pH 7.0 with 0.01 M NaOH using a Crison microTT 2022 automatic titrator. Gastric acid output was expressed as per cent of the output in control rats.
Pharmacological Research, Vol. 22, No. 5, 1990
607
Gastric emptying In the gastric emptying assay, we used a phenol red meal given by oral intubation; the peptides were given at meal time and 60 min later the rats were then sacrificed. The stomach was removed and submitted to the phenol red assay described elsewhere [14]. The results are presented as per cent changes with respect to control rats. The following substances and doses (nmol/rat) were used: EL, 0.06, 0.6 and 6; KS, 0.007, 0.07, 0.7 and 7; PH, 0.6, 6 and 9; SP, 0.06, 0.6 and 6; NKA, 0.08, 0.8 and 8; NKB, 0.06, 0.6 and 3.5; SPT, 1.2 and 11.2; SNK, 0.9, 6 and 9. Data were analysed for statistical significance on an Apple II computer using ANOVA and the Duncan test for post hoc data analysis.
120 SPT
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ao
g
60
~
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• Nt
