- Email: [email protected]
N on mouse colonic propulsion is not affected by mu, kappa, or delta opioid antagonists
anesthesia, a SCS electrode was placed on the dorsal snrtace of the spinal cord (T-12/L1) with the anode placed subcutaneously. After implantation of the SCS electrodes the effect of SCS (50Hz, 02 ms tot 30 rain) on colonic set~sitivity was evaluated In freely moving rats a viscermnotor behavioral response (VMR), quantified as the number of abdominal nmsde contractions was used to determine the level of colonic sensitivity to colorectal distention (CRD) in normal and sensitized colons, Colonic hypersensitivity to irmocuons levels of CRD (30mmHg), was indoced via either a mild irritant acetic acid (0.6%, 1.5 ml) or 30 day's after an acute colitis induced experimentalb/using intracolonic administration of trinitrobenzenesnlfoinc acid (w/v) (TNBS) (50rag&g, 0.5ml, 25% EtOH). Results: tn rats with non-sensitized coIons (n = 6) nc,ctceptive CRD (60 mmHg) increased the VMR from 3 • 0.9 to 25.6 + 3 4 abdominal contractions. Following SCS there was a significant inhibition ot tbe VMR induced by CRD to 5 8 • 2.0 (P
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CRF1 Receptor Mediates Acute and Sustained Visceral Hyperalgesia Following an Acute Stressor in Maternally Separated Long Evans Rats Ines Sehwetz, James A. McRoberts, Santosh V. Coutinho, Sytvie Bradesi, Million Mulugeta, Jerry C Miller, Huping Zhou, Gordon Ohning, Emeran A. Mayer Background: Sustained psychological stress can exacerbate IBS symptoms and stress-induced symptom exacerbation can outlast the actual stressor. Acute, stress-indnced visceral hypersensitivity (SIVH) has been demonstrated in adnh Long Evans rats that were submitted to perinatal maternal separation (Continho et al, 2001). Aims: To determine in maternally separated rats: I) If 1 hour water avoidance eWA) stress has a sustained effect on visceral sensitivity. 2) The role of CRF1 receptors 1 (CRF-1R) in the development of acute and sustained S[VH. Methods: Male pups were separated from the dam for 180 rain daffy from postnatal day 2 to 14 (MS180). Adult rats were equipped with EMG electrodes in abdominal muscle wall. A baseline visceromotor response 0/MR) to graded, phasic colorectal distension (CRD) was determined (10-80 mmHg). After baseline measurement rats were submitted to i hour WA. Immediately after stress and after 24 hours CRD was repeated. A selective CRF1R antagonist, CP 154526 was administered s.c. (32 mg/kg) 45 rain before WA or i hour before 24 hrs measurement. Astressin B (20 microgram&g), a non-selective CRF-R antagonist, was injected intracistemally (i.c.) 15 rain before WA and 15 rain before 24 hrs measurement. The area under the curve (AUC) was determined from each ainmaPs integrated EMG respouse and a ratio was calculated dividing AUC post stress measurement by AUC of baseline. Data represented as mean • SEM Results: Male MS180 rats (n= 11) exhibited significant SIV1-1 immediately after WA (1.29 -+ 0.07) and at 24 hours ( 1.51 +_ 0.13). CP 154526 administered s.c. before WA significantly decreased immediate post WA hyperalgesia compared to vehicle (0.94 -+ 0 0 8 vs. 1.45 • 0.14, p<0.05, n = 11) and tended to decrease delayed hyperalgesia (1.09 -+ 0.18 vs. 2 2 9 0.46, p = 0.09, n = 9). Injection before 24 hrs measurement decreased SWH significantly (0.93 --2- 0.09 vs. 1.2 + 0.06, p<0.05, n=8). Compared to vehide, Astressin B given i.c. abolished immediate (0.84 • 0.14 vs. 1.42 -+ 0,19, p < 0.05) and delayed Vii (1.05 -+ 0.24 vs. 2.23 -+ 0.37, p<0.05), n = 6 in all groups. Condusions: 1) Maternal separation predisposes adult LE rats to devdop acute SPv'H and this effect persists for at least 24 hrs. 2) Both acute and dehyed S I ' ~ are critically dependent on activation of CRF-1R, and presumably increased CRF release during the acute stressor. This study was supported by AstraZeneca R&D Modndal, Sweden and by NIDDK grant 1 P50 0I(64539-01.
W1008 Effect of CNS Administered OFQ/N on Mouse Colonic Propulsion is not Affected by Mu, Kappa, or Delta Opioid Antagonists Robert Kraicbely, Eric A. Gaomnitz, Mark A. Osinski, Paul Bass, Miles L Epstein Background: Og~hanin FQ/Nociceptin (OFQf~), an endogenous opioid-like peptide, has been previously demonstrated to kthibit both colonic and small bowel transit in a routine model througfi both central and peripheral sites (Osinski et al., Am J Physiol, G125-G131, 1999). ghe centrally mediated efiect of OFQ/N on colonic activity is incompletely understood. We investigated the role of CNS and peripheral administration of opioid antagonists on the cemraI effect of OFQ/N. Mefhods: We examined the latenW for expulsion of a 3mm glass bead from the distal colon hallowqng intracerebroventricular 0.c.v.) injection of OFQ/N and ic.v or penpberal administratmn of various opioid antagonists. Results: We observed a dose dependent increase in latency of bead expulsion with i c.v. OF Q.,2,1(1- I0 nmol/monse). The L c v co-administration of OFQ/N with CTOP, nor-binaltorphamine, or naltrindole (mu, kappa, and della antagonists, respectively) did not reduce the bead latency observed with OFQ/N alone Sinrilady, peripheral pretreantrent with the same antagonists did not alter the latency produced by centrally administered OFQ/N. As expected, centrally administered vehicle (saline), as well as the opioid antagonists alone, had no significant effect on bead expuMon when compared to the sham group. Also, as anticipated, Lc.v. administration of the mn agoinsts DAMGO and morphine delayed bead expulsion, an etfect that was blocked by peripheral administration of the opioid receptor antagumst naloxone. Conclusion: We propose that OFQfl'q has an action whose pathway is not influenced by antagonism of mu, kappa, and delta opioid receptors. Tberefore, OFQ/N may represent an important modulator of coloinc propulsion independent ot classical opioid pathways. Supported by NIH grant DK 57018
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Potent Inhibition of Vagal Mechanosenaitivity by Galanin Amanda J. Page, Evelyn Cheng, James Slattery, L A. Black.haw Background. Galanin is a neuropeptide distributed in the peripheral and central ner~o~ system. Its neural actions (mainly inhibitory) may be mediated via 3 subtypes of G-protein coupled receptor. Galanin's effects on somatic sensory fibres may be excitatory or inhibitor'/ and may be mediated via endogenous release (Heppelman et al Eur J Neurosci 12 (2000) 1567). Galanin may also reduce transmission along rags[ afferent fibres from the stomach to the brain stem (Ynan et al JPET 301 (2002) 488) We aimed to determine if gahnin reduces vagal afferent mechanosensitivity in a similar way to that we have found v,~th agonists of other inhibitory G-protein coupled receptors (GABAB and metabotropic glutamate). Methods. The responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli were investigated in vitro before and during application of galanin to their peripheral endings. These afferents were characterized as tension, mncosal and tension/mncosal receptors according to their responsiveness to circumferenual tension (0.5-7g) or mucosal stroki~ (10-1000mg yon Frey hair) or both (Page et al J Physio1512 (1998) 907 ). Results. 2 tension, 3 mncosal and 6 tension/mucosal receptors were tested with human galanin (t0-9 - 108Mr. The responses of all these to tension and stroking were potendy inhibited by gatanin in a concentration dependent manner (ECS0< 10-9M; p<0.05 - p<0.001 two way ANOVA), up to a mammum of 100% inhibition in the 2 tension receptors. Inhibition of mechanosensinvity was reversed after washout of galanin. Selective antagonists are not yet available [or different galanin receptors, although the potency we observed matches the binding characteristics of the GALl receptor (Ki 4.4x10-10M) better than GAL2 (Ki 2.3x10-9M) or GAL3 (Ki 6.9x10-8M) (Branchek et al TIPS 21 (2000) 109). Conclusion. Our data shows a potent inhibitory effect of galanin on gastruesophageal wagal aiferents that is most likely mediated via the GALl receptor subtype. Whether or not endogenous galanin may be involved in regulation of vagal afferent mechanusensitivity is currently under investigation. Supported by NHMRC
WlO09 Role of Kappa- Opioids and Ncurokinin 1 Receptor Interactions on Visceral Nociception in a Rat Model Tobias/.mbregts, Birgit Adam Anion Bertel, Venus Eishabady, Guido Gerken, Gerald Hofimanu gackgnmnd: "fachykinins, like Suhstance P (SP) and opioiderg~c pathways, like kappa opmd receptors modulate visceral sensory fuuction. Very little is known regarding and potential interactions of the receptor system for the regulations of visceral sensory function Aims: (l) to study the effects of a intrathecafly administred (i.th.) selective kappa- opioid agonist and Neurokmin 1 (NK1) antagonists on the visceral sensitivity and (2) to evaluate the interactions of NK1 aud Kappa- opioids receptors in a colorectal distension (CRD) rat model. Methods: Pressure controlled CRD~s (40mmHg) were performed in conscmns male Lewis rats with a barostat inflated ballon catheter and the ~,n~emmotor response (VMR) to CRD in form of abdominal wall contractions was registered by EMG recording of the abdominal wall musculature as baseline measurement(3x phasic 40mmHg CRD) before drug administration and a total of five CRDs 1, 2, 5, 10 and 15 rain after drug application. The selective Kappa opioid receptor agonist U50488 (30nM) and the NK1 antagonist Sendide (S) (5nmol) were gwen alone and in combinalion Lib. into the lumbar spinal cord Results: The kappa agonist caused a decrease of the VMR rangmg from - 12 • 3% after 1rain to a maximum of 35,7 • 5,2% after i5min compared m baseline EMG level. A significant decrease of EMG actMty was also observed after i.th administration of sendide which was only appearant 5 to 15 rain after application ranging from 29,1 • 7,3% to a maramum of 51,1 + 19,8% compared to basefum The combined administration of U50488 and S showed a sigrtificant reduction of the VMR at all time points from 83,8• 13,8% alter lmm m a minimum of 74,4 + 19,9% after 15 iron. Sumn~ary: The kappa agonist and the NK1 antagomst causes a trausiem analgesic etti:ct Coadm inistration of both drugs causes potentiation of the analgesic eftects in the early pMse and an additive dfi-ct during the late response. Conclusion: These data suggest an interaction of kappa opioid receptors and NK1 receptors which might be caused by a kappa mediated SP release. So we conclude that an NKI antagonist could be useful in treatment o f chronic visceral pain because of its analgesic etfect and the potentiation of centrally released uDmds • • "+
AGA Abstracts
W1012
Central human Uroeortin-ll (h Ucn II) and h/rat CRF delay gastric emptying through distinct neural pathways in rats Jozsef Czimmer, Mulugela Million, Yvette ]'ache Background: CRF and urocortin injected intracistemally tic) act within the brain to delay gastric emptying and motility, CRF and urocortin actions are both blocked by the selectb~ CRF2 receptor antagonist, astressin2-B, while the selective CRFI antagonist has no effect (Brain Res. 893:29-35, 2001; BJP 136:237-247, 2002). Human Urocortin-I1 (Ucn II), isa new member of the CRF family with selective affinity to the CRF2 receptor (PNAS 98:2g 43-48, 2001). Aim: To characterize the central action of hUcn II on gastric motor function and compare it with that of h/rCRF in rats. Methods: SD rats were injected ic with either Uen II (0.1 or 1 ~g), CRF (0.3 or 1 ~g) or vehicle tic, 10 ~l). Astressin2-B tic, 5 ~l) 0r vehicle was injected just before; subdiaphragmatic vagotomy was performed 48 h before; and bretyliom tosyiate (25 mg/kg) was injected ip 60 rain before ic CRF or Ucn If. Semiliquid gastric emptying (GE) was determined 20 toni after oro-gastric garage of 15% methykdhlose-phenol red solution 15 rain after ic CRF or Ucn I1. Results: CRF (0.3 or 1 tzg) decreased significantly GE to * 23.9-+ 5.2%, and *9.6 • 3.6%, and ic Ucn l! (0.1 or 1 ~g) to "37.7_+ 68% and "23.1 +8.5% respectively compared to control group (61.0"+4.3%). Astressin2-B (3 I~g, ic) did not influence basal GE (543 • 7.2%) and antagonized both CRF and Ucn II action (GE: 64.1 + 4.3% and 51 6 -+ 2 8% respectively compared with vehicle: 54.4_+ 8.5%).
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CRF1 Receptor Mediates Acute and Sustained Visceral Hyperalgesia Following an Acute Stressor in Maternally Separated Long Evans Rats Ines Sehwetz, James A. McRoberts, Santosh V. Coutinho, Sytvie Bradesi, Million Mulugeta, Jerry C Miller, Huping Zhou, Gordon Ohning, Emeran A. Mayer Background: Sustained psychological stress can exacerbate IBS symptoms and stress-induced symptom exacerbation can outlast the actual stressor. Acute, stress-indnced visceral hypersensitivity (SIVH) has been demonstrated in adnh Long Evans rats that were submitted to perinatal maternal separation (Continho et al, 2001). Aims: To determine in maternally separated rats: I) If 1 hour water avoidance eWA) stress has a sustained effect on visceral sensitivity. 2) The role of CRF1 receptors 1 (CRF-1R) in the development of acute and sustained S[VH. Methods: Male pups were separated from the dam for 180 rain daffy from postnatal day 2 to 14 (MS180). Adult rats were equipped with EMG electrodes in abdominal muscle wall. A baseline visceromotor response 0/MR) to graded, phasic colorectal distension (CRD) was determined (10-80 mmHg). After baseline measurement rats were submitted to i hour WA. Immediately after stress and after 24 hours CRD was repeated. A selective CRF1R antagonist, CP 154526 was administered s.c. (32 mg/kg) 45 rain before WA or i hour before 24 hrs measurement. Astressin B (20 microgram&g), a non-selective CRF-R antagonist, was injected intracistemally (i.c.) 15 rain before WA and 15 rain before 24 hrs measurement. The area under the curve (AUC) was determined from each ainmaPs integrated EMG respouse and a ratio was calculated dividing AUC post stress measurement by AUC of baseline. Data represented as mean • SEM Results: Male MS180 rats (n= 11) exhibited significant SIV1-1 immediately after WA (1.29 -+ 0.07) and at 24 hours ( 1.51 +_ 0.13). CP 154526 administered s.c. before WA significantly decreased immediate post WA hyperalgesia compared to vehicle (0.94 -+ 0 0 8 vs. 1.45 • 0.14, p<0.05, n = 11) and tended to decrease delayed hyperalgesia (1.09 -+ 0.18 vs. 2 2 9 0.46, p = 0.09, n = 9). Injection before 24 hrs measurement decreased SWH significantly (0.93 --2- 0.09 vs. 1.2 + 0.06, p<0.05, n=8). Compared to vehide, Astressin B given i.c. abolished immediate (0.84 • 0.14 vs. 1.42 -+ 0,19, p < 0.05) and delayed Vii (1.05 -+ 0.24 vs. 2.23 -+ 0.37, p<0.05), n = 6 in all groups. Condusions: 1) Maternal separation predisposes adult LE rats to devdop acute SPv'H and this effect persists for at least 24 hrs. 2) Both acute and dehyed S I ' ~ are critically dependent on activation of CRF-1R, and presumably increased CRF release during the acute stressor. This study was supported by AstraZeneca R&D Modndal, Sweden and by NIDDK grant 1 P50 0I(64539-01.
W1008 Effect of CNS Administered OFQ/N on Mouse Colonic Propulsion is not Affected by Mu, Kappa, or Delta Opioid Antagonists Robert Kraicbely, Eric A. Gaomnitz, Mark A. Osinski, Paul Bass, Miles L Epstein Background: Og~hanin FQ/Nociceptin (OFQf~), an endogenous opioid-like peptide, has been previously demonstrated to kthibit both colonic and small bowel transit in a routine model througfi both central and peripheral sites (Osinski et al., Am J Physiol, G125-G131, 1999). ghe centrally mediated efiect of OFQ/N on colonic activity is incompletely understood. We investigated the role of CNS and peripheral administration of opioid antagonists on the cemraI effect of OFQ/N. Mefhods: We examined the latenW for expulsion of a 3mm glass bead from the distal colon hallowqng intracerebroventricular 0.c.v.) injection of OFQ/N and ic.v or penpberal administratmn of various opioid antagonists. Results: We observed a dose dependent increase in latency of bead expulsion with i c.v. OF Q.,2,1(1- I0 nmol/monse). The L c v co-administration of OFQ/N with CTOP, nor-binaltorphamine, or naltrindole (mu, kappa, and della antagonists, respectively) did not reduce the bead latency observed with OFQ/N alone Sinrilady, peripheral pretreantrent with the same antagonists did not alter the latency produced by centrally administered OFQ/N. As expected, centrally administered vehicle (saline), as well as the opioid antagonists alone, had no significant effect on bead expuMon when compared to the sham group. Also, as anticipated, Lc.v. administration of the mn agoinsts DAMGO and morphine delayed bead expulsion, an etfect that was blocked by peripheral administration of the opioid receptor antagumst naloxone. Conclusion: We propose that OFQfl'q has an action whose pathway is not influenced by antagonism of mu, kappa, and delta opioid receptors. Tberefore, OFQ/N may represent an important modulator of coloinc propulsion independent ot classical opioid pathways. Supported by NIH grant DK 57018
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Potent Inhibition of Vagal Mechanosenaitivity by Galanin Amanda J. Page, Evelyn Cheng, James Slattery, L A. Black.haw Background. Galanin is a neuropeptide distributed in the peripheral and central ner~o~ system. Its neural actions (mainly inhibitory) may be mediated via 3 subtypes of G-protein coupled receptor. Galanin's effects on somatic sensory fibres may be excitatory or inhibitor'/ and may be mediated via endogenous release (Heppelman et al Eur J Neurosci 12 (2000) 1567). Galanin may also reduce transmission along rags[ afferent fibres from the stomach to the brain stem (Ynan et al JPET 301 (2002) 488) We aimed to determine if gahnin reduces vagal afferent mechanosensitivity in a similar way to that we have found v,~th agonists of other inhibitory G-protein coupled receptors (GABAB and metabotropic glutamate). Methods. The responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli were investigated in vitro before and during application of galanin to their peripheral endings. These afferents were characterized as tension, mncosal and tension/mncosal receptors according to their responsiveness to circumferenual tension (0.5-7g) or mucosal stroki~ (10-1000mg yon Frey hair) or both (Page et al J Physio1512 (1998) 907 ). Results. 2 tension, 3 mncosal and 6 tension/mucosal receptors were tested with human galanin (t0-9 - 108Mr. The responses of all these to tension and stroking were potendy inhibited by gatanin in a concentration dependent manner (ECS0< 10-9M; p<0.05 - p<0.001 two way ANOVA), up to a mammum of 100% inhibition in the 2 tension receptors. Inhibition of mechanosensinvity was reversed after washout of galanin. Selective antagonists are not yet available [or different galanin receptors, although the potency we observed matches the binding characteristics of the GALl receptor (Ki 4.4x10-10M) better than GAL2 (Ki 2.3x10-9M) or GAL3 (Ki 6.9x10-8M) (Branchek et al TIPS 21 (2000) 109). Conclusion. Our data shows a potent inhibitory effect of galanin on gastruesophageal wagal aiferents that is most likely mediated via the GALl receptor subtype. Whether or not endogenous galanin may be involved in regulation of vagal afferent mechanusensitivity is currently under investigation. Supported by NHMRC
WlO09 Role of Kappa- Opioids and Ncurokinin 1 Receptor Interactions on Visceral Nociception in a Rat Model Tobias/.mbregts, Birgit Adam Anion Bertel, Venus Eishabady, Guido Gerken, Gerald Hofimanu gackgnmnd: "fachykinins, like Suhstance P (SP) and opioiderg~c pathways, like kappa opmd receptors modulate visceral sensory fuuction. Very little is known regarding and potential interactions of the receptor system for the regulations of visceral sensory function Aims: (l) to study the effects of a intrathecafly administred (i.th.) selective kappa- opioid agonist and Neurokmin 1 (NK1) antagonists on the visceral sensitivity and (2) to evaluate the interactions of NK1 aud Kappa- opioids receptors in a colorectal distension (CRD) rat model. Methods: Pressure controlled CRD~s (40mmHg) were performed in conscmns male Lewis rats with a barostat inflated ballon catheter and the ~,n~emmotor response (VMR) to CRD in form of abdominal wall contractions was registered by EMG recording of the abdominal wall musculature as baseline measurement(3x phasic 40mmHg CRD) before drug administration and a total of five CRDs 1, 2, 5, 10 and 15 rain after drug application. The selective Kappa opioid receptor agonist U50488 (30nM) and the NK1 antagonist Sendide (S) (5nmol) were gwen alone and in combinalion Lib. into the lumbar spinal cord Results: The kappa agonist caused a decrease of the VMR rangmg from - 12 • 3% after 1rain to a maximum of 35,7 • 5,2% after i5min compared m baseline EMG level. A significant decrease of EMG actMty was also observed after i.th administration of sendide which was only appearant 5 to 15 rain after application ranging from 29,1 • 7,3% to a maramum of 51,1 + 19,8% compared to basefum The combined administration of U50488 and S showed a sigrtificant reduction of the VMR at all time points from 83,8• 13,8% alter lmm m a minimum of 74,4 + 19,9% after 15 iron. Sumn~ary: The kappa agonist and the NK1 antagomst causes a trausiem analgesic etti:ct Coadm inistration of both drugs causes potentiation of the analgesic eftects in the early pMse and an additive dfi-ct during the late response. Conclusion: These data suggest an interaction of kappa opioid receptors and NK1 receptors which might be caused by a kappa mediated SP release. So we conclude that an NKI antagonist could be useful in treatment o f chronic visceral pain because of its analgesic etfect and the potentiation of centrally released uDmds • • "+
AGA Abstracts
W1012
Central human Uroeortin-ll (h Ucn II) and h/rat CRF delay gastric emptying through distinct neural pathways in rats Jozsef Czimmer, Mulugela Million, Yvette ]'ache Background: CRF and urocortin injected intracistemally tic) act within the brain to delay gastric emptying and motility, CRF and urocortin actions are both blocked by the selectb~ CRF2 receptor antagonist, astressin2-B, while the selective CRFI antagonist has no effect (Brain Res. 893:29-35, 2001; BJP 136:237-247, 2002). Human Urocortin-I1 (Ucn II), isa new member of the CRF family with selective affinity to the CRF2 receptor (PNAS 98:2g 43-48, 2001). Aim: To characterize the central action of hUcn II on gastric motor function and compare it with that of h/rCRF in rats. Methods: SD rats were injected ic with either Uen II (0.1 or 1 ~g), CRF (0.3 or 1 ~g) or vehicle tic, 10 ~l). Astressin2-B tic, 5 ~l) 0r vehicle was injected just before; subdiaphragmatic vagotomy was performed 48 h before; and bretyliom tosyiate (25 mg/kg) was injected ip 60 rain before ic CRF or Ucn If. Semiliquid gastric emptying (GE) was determined 20 toni after oro-gastric garage of 15% methykdhlose-phenol red solution 15 rain after ic CRF or Ucn I1. Results: CRF (0.3 or 1 tzg) decreased significantly GE to * 23.9-+ 5.2%, and *9.6 • 3.6%, and ic Ucn l! (0.1 or 1 ~g) to "37.7_+ 68% and "23.1 +8.5% respectively compared to control group (61.0"+4.3%). Astressin2-B (3 I~g, ic) did not influence basal GE (543 • 7.2%) and antagonized both CRF and Ucn II action (GE: 64.1 + 4.3% and 51 6 -+ 2 8% respectively compared with vehicle: 54.4_+ 8.5%).
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