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Sensitivity of mesenteric afferents to vascular perfusion in rat ileum in vitro
GASTROENTEROLOGY Vol. 118, No.4
A380 AGA ABSTRACTS
2007
2009
SOMATOSTATIN SST 2 RECEPTOR AGONISTS INHIBIT BRADYKININ-SENSITIVE JEJUNAL AFFERENTS IN THE RAT. Charlotte E. Booth, Anthony 1. Kirkup, Gareth A. Hicks, Patrick P. Humphrey, David Grundy, Univ of Sheffield, Sheffield, United Kingdom; Glaxo Institute of Applied Pharmacology, Cambridge, United Kingdom. Visceral hypersensitivity has been recognized in some patients with IBS. A reduction in visceral sensitivity by somatostatin receptor agonists in humans (Hasler et al, '93 Gastroenterol. 104, 1390-7) may arise from a peripheral action on sensory signal transduction. However, the mechanisms underlying this effect and the receptors involved are unknown. The aim of the present study was therefore to examine the effect of octreotide and the selective sst, receptor agonist, BIM 23027 (BIM) on high threshold mechano- and bradykinin-sensitive jejunal afferents in the rat. Extracellular recordings of jejunal afferent nerve activity were obtained from pentobarbitone-anaesthetized (60mglkg, i.p.) male Wistar rats (330-450g) as previously described (Kirkup et al, '99 J. Physiol. 520, 277). Data are the mean z s.e.m from 4-14 experiments, analyzed using the Rank-sum test. BIM (lOOp,glkg, i.v.) inhibited the high threshold afferent response to ramp distension (0-60mmHg, Imllmin saline infusion). Significant inhibition (approx 25%) was observed at distending pressures between 20 & 45mmHg. BIM (1-100 p,glkg i.v.) induced a dose-dependent inhibition of the BK-evoked (0.1-1 p,glkg i.a.) afferent excitation. The degree of inhibition was dependent upon both BK and BIM dose and was similar in extent to that observed with octreotide (Table 1). In the absence of applied sst, receptor agonists, the selective sstz antagonist, Cyanamid 154806 (lmglkg i.v.), augmented the BK- (O.lp,glkg) evoked excitation by 63±% (p<0.05, n =4) suggesting that in the anaesthetized rat, endogenous somatostatin may regulate afferent sensitivity. In conclusion, these data suggest that activation of somatostatin sst, receptors inhibits populations of mesenteric afferent nerves likely to be involved in nociceptive transmission.
DIFFERENTIAL INVOLVEMENT OF SPINAL NMDA, CGRP AND NKI RECEPTORS IN RESPONSES TO PHASIC VS. TONIC COLORECTAL DISTENSION. Santosh V. Coutinho, Jerry C. Miller, Kerstin Hubel, Alfred I. Bayati, Gerald Holtmann, Emeran A. Mayer, Ucla/Cure, Los Angeles, CA; Univ of Essen, Essen, Germany; AstraZeneca, Molndal, Sweden. We have recently observed that unlike brief, phasic colorectal distension(CRD), prolonged noxious CRD leads to an enhanced response to a subsequent tonic stimulus. AIM: To ascertain the involvement of spinal NMDA, CGRP and NKI receptors in modulating responses to brief, phasic vs. prolonged, tonic CRD in unanesthetized rats. METHODS: Experiments were performed on male Sprague-Dawley rats. CRD was achieved by pressure-controlled air inflation of a latex balloon positioned I em proximal to the anus. The visceromotor response (VMR) evoked by CRD was quantified by either visual inspection of abdominal contractions (in a blinded manner) or by monitoring electromyographic (EMG) activity in the external oblique musculature. The NMDA receptor open channel blocker, MK-801 (10 nmol), or the CGRP receptor antagonist, hCGRP8-37 (10 nmol), or the NKI receptor antagonist, RP67580 (10 nmol), or the vehicle, saline, was administered directly to the lumbar spinal cord through a chronically implanted indwelling intrathecal (i.t.) catheter. Results: Animals that received noxious, tonic CRD (60 mmHg, 10 min duration, 16 min interstimulus interval) exhibited a significant increase in the magnitude of the VMR to the second tonic stimulus. This sensitization appeared to be short-lived and response magnitudes reverted to control levels by the fourth trial. In contrast, the VMR to phasic CRD (60 mmHg, 20 s duration, 4 min interstimulus interval) was stable with time. All three antagonists, but not saline, administered i.t. 10 min prior to the first tonic stimulus produced a significant attenuation of the responses to the first as well as the second tonic stimulus. All three antagonists, but not saline, administered i.t, 10 min prior to the second tonic stimulus not only prevented sensitization, but also significantly attenuated the magnitude of the response to the second tonic stimulus (compared to the first). In contrast, all three antagonists were without effect on the VMR to phasic CRD. Conclusions: These data suggest that spinal NMDA receptors, CGRP receptors and NKI receptors playa role in modulating the response to tonic CRD, but are not involved in responses to phasic CRD. Additionally, it appears that while prolonged tonic CRD, and likely contractions, can induce central sensitization, phasic events (even in the noxious range) cannot. This may provide a plausible explanation for the induction of visceral hyperalgesia by certain tonic motility events in the gut.
Table 1:Effect ofoctreotide and 81M 23027 ontheafferent responses tobradykinin. % ofcontrol responses Octreotide ftg.kg·' (i.v.) BIM 23027 ft!!.kg· (i.v.) '
0.1l!g.kg·1 BK(l.a.) 0.3l!g.kg· ' BK(i.a.) 1l!g.kg·' BK (i.a.)
10
100
10
100
111.9:t57
535:t11.9
33.1:t9.5
105.3:t7.4
649:t51
32.0:t76
100.5:t6.7
575±11.2
2B.1±B.7
106.3:t5.B
69.1:t5.B
40.3:t4.9
997±4.0
92.1±22.5
41.9:t9.4
10B.B:t9.l
79.4:t6.3
55.0±11.9
2008 SENSITIVITY OF MESENTERIC AFFERENTS TO VASCULAR PERFUSION IN RAT ILEUM IN VITRO. Alan M. Brunsden, Samuel Jacob, Kama Dev Bardhan, David Grundy, Sheffield Univ, Sheffield, United Kingdom; Rotherham Gen Hosp, Rotherham, United Kingdom. Introduction: Gastrointestinal arterioles are richly innervated by extrinsic sensory nerves which play an important role in control of GI blood flow. However, the ability of these afferents to 'sense' vascular flow is unknown. We have developed a novel in vitro preparation to investigate this possibility. Method: Segments of terminal ileum (from anaesthetised rats)were perfused serosally (5mllmin) with gassed bicarbonate saline (BS), luminally with saline (1.0ml/min) and arterially with BS (+3% dextran,1OmM glucose,0.6mM glutamine). Extracellular recordings were made from paravascular nerves in an adjacent chamber. Vascular perfusion pressure (VPP), intraluminal pressure (lLP) and afferent discharge (AD) were monitored simultaneously. AD is expressed as total impulses (area under the response), effects on VPP calculated as % changes, and motility measured as area under spontaneous contractions. Data (mean :t S.E.M.) were compared using Mann-Whitney and Dunns multiple comparison tests after ANOV A, *p<0.05). Results: Increasing vascular flow rate step-wise for 5 min from 0.6 ml/min to 1.0,1.4 and 1.8 mllmin caused a concomitant reduction in baseline AD in 6/8 studies. Discharge fell to 79 :t 8%, 68 :t 9%, and 59 :t 13% of basal respectively, returning to 96 :t 23% upon perfusion at 0.6mllmin. In addition, stopping flow for 5 min increased AD dramatically in all 8 experiments (244 :t 44 % of basal, p<0.05) and returned to basal levels upon re-perfusion at 0.6mllmin (112 :t 14%, p>0.05). The step-wise increase in flow rate also appeared to increase motility and contractions were markedly attenuated when flow was stopped (l66:t 51 to 16 :t 8 cmll-O, p<0.05). In segments perfused vascularly at Imllmin and pre-constricted with serosal L-phenylephrine (lOOp,M),luminal capsaicin (2ml, 100J-tM) caused a transient (73 :t 14 s duration) activation of AD (1652 :t 447 impulses) compared to vehicle controls (42:t 15 impulses p<0.05), and this was accompanied (after 51 :t 22s) by a sustained reduction in VPP (capsaicin, -6.1 :t 1.9% vs -0.15 :t O.I,control, p<0.05) after 5.5 :!:: 0.4 min. This was indedpendent of changes in ILP(capsaicin, 25 :t 7 vs 28 :!:: 4 cmHzO, control, p>0.05). Conclusions: These observations support a role for extrinsic afferents in regulating intestinal vascular tone. However, afferents may in tum detect changes in vascular perfusion. Whether this represents direct 'cross-talk' between afferents and vasculature or an indirect response to local factors remains to be determined. Supported by Bardhan Research & Education Trust
2010 PROSTAGLANDIN E z ALTERS CALCIUM HOMEOSTASIS IN RAT VISCERAL SENSORY NEURONS. Joseph R. Evans, Helgi K. Sigmundsson, Klaus Bielefeldt, Univ of Iowa, Iowa City, IA. Prostanoids play an important role in altering cellular function during inflammation. We hypothesized that prostaglandin Ez (PGE2) may change visceral sensation by directly affecting primary sensory neurons. METHODS: Rat nodose neurons were dissociated and cultured for 24 h prior to loading with the calcium indicator fura-2-AM. The intracellular calcium concentration ([CaH ]) was measured using a videofluorescence imaging system. RESULTS: PGE2 (I p,M) caused a slow rise in [Ca2+] in 11/30 (37%) cells (figure) which peaked at 42.9:t8.3 % over baseline (p
Sample trace demonstrating the effect of PGE2
Time(s)
A380 AGA ABSTRACTS
2007
2009
SOMATOSTATIN SST 2 RECEPTOR AGONISTS INHIBIT BRADYKININ-SENSITIVE JEJUNAL AFFERENTS IN THE RAT. Charlotte E. Booth, Anthony 1. Kirkup, Gareth A. Hicks, Patrick P. Humphrey, David Grundy, Univ of Sheffield, Sheffield, United Kingdom; Glaxo Institute of Applied Pharmacology, Cambridge, United Kingdom. Visceral hypersensitivity has been recognized in some patients with IBS. A reduction in visceral sensitivity by somatostatin receptor agonists in humans (Hasler et al, '93 Gastroenterol. 104, 1390-7) may arise from a peripheral action on sensory signal transduction. However, the mechanisms underlying this effect and the receptors involved are unknown. The aim of the present study was therefore to examine the effect of octreotide and the selective sst, receptor agonist, BIM 23027 (BIM) on high threshold mechano- and bradykinin-sensitive jejunal afferents in the rat. Extracellular recordings of jejunal afferent nerve activity were obtained from pentobarbitone-anaesthetized (60mglkg, i.p.) male Wistar rats (330-450g) as previously described (Kirkup et al, '99 J. Physiol. 520, 277). Data are the mean z s.e.m from 4-14 experiments, analyzed using the Rank-sum test. BIM (lOOp,glkg, i.v.) inhibited the high threshold afferent response to ramp distension (0-60mmHg, Imllmin saline infusion). Significant inhibition (approx 25%) was observed at distending pressures between 20 & 45mmHg. BIM (1-100 p,glkg i.v.) induced a dose-dependent inhibition of the BK-evoked (0.1-1 p,glkg i.a.) afferent excitation. The degree of inhibition was dependent upon both BK and BIM dose and was similar in extent to that observed with octreotide (Table 1). In the absence of applied sst, receptor agonists, the selective sstz antagonist, Cyanamid 154806 (lmglkg i.v.), augmented the BK- (O.lp,glkg) evoked excitation by 63±% (p<0.05, n =4) suggesting that in the anaesthetized rat, endogenous somatostatin may regulate afferent sensitivity. In conclusion, these data suggest that activation of somatostatin sst, receptors inhibits populations of mesenteric afferent nerves likely to be involved in nociceptive transmission.
DIFFERENTIAL INVOLVEMENT OF SPINAL NMDA, CGRP AND NKI RECEPTORS IN RESPONSES TO PHASIC VS. TONIC COLORECTAL DISTENSION. Santosh V. Coutinho, Jerry C. Miller, Kerstin Hubel, Alfred I. Bayati, Gerald Holtmann, Emeran A. Mayer, Ucla/Cure, Los Angeles, CA; Univ of Essen, Essen, Germany; AstraZeneca, Molndal, Sweden. We have recently observed that unlike brief, phasic colorectal distension(CRD), prolonged noxious CRD leads to an enhanced response to a subsequent tonic stimulus. AIM: To ascertain the involvement of spinal NMDA, CGRP and NKI receptors in modulating responses to brief, phasic vs. prolonged, tonic CRD in unanesthetized rats. METHODS: Experiments were performed on male Sprague-Dawley rats. CRD was achieved by pressure-controlled air inflation of a latex balloon positioned I em proximal to the anus. The visceromotor response (VMR) evoked by CRD was quantified by either visual inspection of abdominal contractions (in a blinded manner) or by monitoring electromyographic (EMG) activity in the external oblique musculature. The NMDA receptor open channel blocker, MK-801 (10 nmol), or the CGRP receptor antagonist, hCGRP8-37 (10 nmol), or the NKI receptor antagonist, RP67580 (10 nmol), or the vehicle, saline, was administered directly to the lumbar spinal cord through a chronically implanted indwelling intrathecal (i.t.) catheter. Results: Animals that received noxious, tonic CRD (60 mmHg, 10 min duration, 16 min interstimulus interval) exhibited a significant increase in the magnitude of the VMR to the second tonic stimulus. This sensitization appeared to be short-lived and response magnitudes reverted to control levels by the fourth trial. In contrast, the VMR to phasic CRD (60 mmHg, 20 s duration, 4 min interstimulus interval) was stable with time. All three antagonists, but not saline, administered i.t. 10 min prior to the first tonic stimulus produced a significant attenuation of the responses to the first as well as the second tonic stimulus. All three antagonists, but not saline, administered i.t, 10 min prior to the second tonic stimulus not only prevented sensitization, but also significantly attenuated the magnitude of the response to the second tonic stimulus (compared to the first). In contrast, all three antagonists were without effect on the VMR to phasic CRD. Conclusions: These data suggest that spinal NMDA receptors, CGRP receptors and NKI receptors playa role in modulating the response to tonic CRD, but are not involved in responses to phasic CRD. Additionally, it appears that while prolonged tonic CRD, and likely contractions, can induce central sensitization, phasic events (even in the noxious range) cannot. This may provide a plausible explanation for the induction of visceral hyperalgesia by certain tonic motility events in the gut.
Table 1:Effect ofoctreotide and 81M 23027 ontheafferent responses tobradykinin. % ofcontrol responses Octreotide ftg.kg·' (i.v.) BIM 23027 ft!!.kg· (i.v.) '
0.1l!g.kg·1 BK(l.a.) 0.3l!g.kg· ' BK(i.a.) 1l!g.kg·' BK (i.a.)
10
100
10
100
111.9:t57
535:t11.9
33.1:t9.5
105.3:t7.4
649:t51
32.0:t76
100.5:t6.7
575±11.2
2B.1±B.7
106.3:t5.B
69.1:t5.B
40.3:t4.9
997±4.0
92.1±22.5
41.9:t9.4
10B.B:t9.l
79.4:t6.3
55.0±11.9
2008 SENSITIVITY OF MESENTERIC AFFERENTS TO VASCULAR PERFUSION IN RAT ILEUM IN VITRO. Alan M. Brunsden, Samuel Jacob, Kama Dev Bardhan, David Grundy, Sheffield Univ, Sheffield, United Kingdom; Rotherham Gen Hosp, Rotherham, United Kingdom. Introduction: Gastrointestinal arterioles are richly innervated by extrinsic sensory nerves which play an important role in control of GI blood flow. However, the ability of these afferents to 'sense' vascular flow is unknown. We have developed a novel in vitro preparation to investigate this possibility. Method: Segments of terminal ileum (from anaesthetised rats)were perfused serosally (5mllmin) with gassed bicarbonate saline (BS), luminally with saline (1.0ml/min) and arterially with BS (+3% dextran,1OmM glucose,0.6mM glutamine). Extracellular recordings were made from paravascular nerves in an adjacent chamber. Vascular perfusion pressure (VPP), intraluminal pressure (lLP) and afferent discharge (AD) were monitored simultaneously. AD is expressed as total impulses (area under the response), effects on VPP calculated as % changes, and motility measured as area under spontaneous contractions. Data (mean :t S.E.M.) were compared using Mann-Whitney and Dunns multiple comparison tests after ANOV A, *p<0.05). Results: Increasing vascular flow rate step-wise for 5 min from 0.6 ml/min to 1.0,1.4 and 1.8 mllmin caused a concomitant reduction in baseline AD in 6/8 studies. Discharge fell to 79 :t 8%, 68 :t 9%, and 59 :t 13% of basal respectively, returning to 96 :t 23% upon perfusion at 0.6mllmin. In addition, stopping flow for 5 min increased AD dramatically in all 8 experiments (244 :t 44 % of basal, p<0.05) and returned to basal levels upon re-perfusion at 0.6mllmin (112 :t 14%, p>0.05). The step-wise increase in flow rate also appeared to increase motility and contractions were markedly attenuated when flow was stopped (l66:t 51 to 16 :t 8 cmll-O, p<0.05). In segments perfused vascularly at Imllmin and pre-constricted with serosal L-phenylephrine (lOOp,M),luminal capsaicin (2ml, 100J-tM) caused a transient (73 :t 14 s duration) activation of AD (1652 :t 447 impulses) compared to vehicle controls (42:t 15 impulses p<0.05), and this was accompanied (after 51 :t 22s) by a sustained reduction in VPP (capsaicin, -6.1 :t 1.9% vs -0.15 :t O.I,control, p<0.05) after 5.5 :!:: 0.4 min. This was indedpendent of changes in ILP(capsaicin, 25 :t 7 vs 28 :!:: 4 cmHzO, control, p>0.05). Conclusions: These observations support a role for extrinsic afferents in regulating intestinal vascular tone. However, afferents may in tum detect changes in vascular perfusion. Whether this represents direct 'cross-talk' between afferents and vasculature or an indirect response to local factors remains to be determined. Supported by Bardhan Research & Education Trust
2010 PROSTAGLANDIN E z ALTERS CALCIUM HOMEOSTASIS IN RAT VISCERAL SENSORY NEURONS. Joseph R. Evans, Helgi K. Sigmundsson, Klaus Bielefeldt, Univ of Iowa, Iowa City, IA. Prostanoids play an important role in altering cellular function during inflammation. We hypothesized that prostaglandin Ez (PGE2) may change visceral sensation by directly affecting primary sensory neurons. METHODS: Rat nodose neurons were dissociated and cultured for 24 h prior to loading with the calcium indicator fura-2-AM. The intracellular calcium concentration ([CaH ]) was measured using a videofluorescence imaging system. RESULTS: PGE2 (I p,M) caused a slow rise in [Ca2+] in 11/30 (37%) cells (figure) which peaked at 42.9:t8.3 % over baseline (p
Sample trace demonstrating the effect of PGE2
Time(s)