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Is ineffective peristalsis really ineffective?
Sl137
may be different from those mediating sustained contraction, which may depend on a Rhokinase pathway. We therefore tested the proposition that sustained LES tone, which depends on elevated levels of endogenous PGF2a may be maintained through a Rho-kinase dependent pathway. In LES circular muscle strips the Rho-kinase inhibitor Y27632 dose-dependently decreased tone and abolished it at 10 ~ M, suggesting that LES tone is Rho-kinase- dependent. The PGF2a antagonist AL-8810 dose-dependently reduced tone. In strips treated with indomethacin to abolish production of endogenous PGF2a, exogenously administered PGF2a maintained a sustained contraction lasting more than 30 rain. The sustained, but not the initial contraction in response to PGF2a was abolished by Y27632. LES circular smooth muscle cells, isolated by enzymatic digestion were used to exanaine the intracelhilar contractile pathways mediating Rho-kinase- PGF2a-dependent sustained contraction. In cells PGF2a caused a relatively rapid contraction that achieved maximum shortening in approximately 30 seconds and persisted in excess of 20 minutes. After treatment with the Rho-kinase inhibitor Y27632 cells achieved the same maximum initial shortening as untreated ceils, but did not remain contracted, and returned to their unstimulated length within approximately 10 minutes. The inhibition of sustained contraction by Y27632 in cells mimics the inhibition of tone and of sustained PGF2a-induced contraction in strips In saponin-permeabilized cells, PGF2a-induced shortening was the same as in intact ceils. In these permeable cells G~3 and RhoA antibodies reduced and the selective RhoA inhibitor C3 (exoenzyme of Clostridium Botulinum) abolished sustained contraction induced by PGF2a, confirming that the contraction is mediated by RhoA-induced activation of Rhokinase. The data suggest a role of RhoA- Rho-Kinase in maintenance of PGF2a induced sustained contraction and LES tone. Supported by NIH ROI-DK-28614.
Not Just a Failure to Relax: Timing and Magnitude of Lower Esophageal Sphincter After-Contractions Also Contribute to Esophageal Outflow Obstruction in Achalasia Samer Gawrieh, Michael C. Jean, Benson T. Massey BACKGROUND: Impaired esophageal emptying is a cardinal finding in achalasia and is traditionally felt to result from failed lower esophageal sphincter (LES) relaxation and absent esophageal body peristalsis, little attention has been given to the presence of deglutitive after-conrtactions (ACs) in achalasia and their possible effect on esophageal emptying. METHODS: Manomet~ac recordings from 68 achalasia patients (36 F, mean 47 115-85] yr) referred to our clinical manometry laboratory were analyzed. Studies were performed with the subjects lasting and supine, using a sleeve device to record from the LES. The following parameters were scored for three 5ml water swallows: Initial LES pressure (LESP); time (from swallow onset) and magnitude of peak esophageal body pressure (EBP) wave (located 2 cm above the top of the LES); incidence, time, and magnitude of LES deglutitive AC; maximum value by which EBP exceeded LESP during any time after the swallow; basal EflP between swallows. In addition, whether EBP waveforms were isobaric or non-isobaric was recorded. Values were compared to those in 16 subjects (8F, mean 47 [14-881 yr) with normal peristalsis and LES relaxation. RESULTS: The incidence of LES AC was similar in achalasia and controls (84% vs. 92%). As expected, basal LESP was higher (31 vs. 22 mmHg, p<.001) and peak EBP was lower (34 vs. 93 mmHg, p<.001) in achalasia. In achalasia, time of peak EPB was earlier (4.5 vs. 8.2 s, p<.O01) and peak EBP generally exceeded basal LESP (p<.001). However, LES ACs also occurred earlier in achalasia (5.8 vs. 9.8 s, p<.001) and typically exceeded peak EBP (55 mmHg vs. 34 mmHg, p<.O01). As a result, the EBPLESP differential did not favor emptying in achalasia, whereas it did in controls (-4 vs. 13 mmHg, p<.001). The worse this differential, the higher the basal EBP was in achalasia patients (p= .008). Swallows showing non-isobaric waveforms tended to have a favorable pressure differential compared to those that were isobaric (3 vs. -4 mmHg, p = .026). In a multiple regression analysis, factors sigraficantly determining the pressure differential were: peak EBP (positively); basal LESP, time of LES AC and magnitude of LES AC (negatively). Higher peak EBP were offset by their association with higher LES AC (p<.001). CONCLUSIONS: In achalasia, esophageal outflow obstruction is not simply a matter of the LES not relaxing. Timing and magnitude of LES ACs also contribute to this obstruction. Supported by NIH PO1 03191-03.
Sl140 Integrin Linked Kinase (ILK) in ACh-lnduced Contraction of Cat Esophageal Circular Smooth Muscle (ESO) Weibiao Cao, Ling Chang, Karen M. Hamett, .ling T. Dang, Jose Behar, Michael P. Walsh, Piero Biancani We have previously shown that ACh-induced contraction of cat ESO is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid, activation of the Cat§ -insensitive protein kinase C-~ (PKCE) and activation of two distinct MAP kinase pathways: an ERK1/ERK2 MAP kinase-dependent pathway and an HSP27-1inked p38 kinasedependent pathway. The sequence of events occurring between activation of MAP kinases and muscle contraction, however, has not been elucidated. A possible candidate intermediate protein in Ca2+-independent PKCt-mediated contraction may be ILK, a kinase associated with myofilaments and responsible for Ca2§ -independent phnsphorylation of smooth muscle myosin. We therefore examined the role of ILK in ACh-induced contraction of cat ESO. ILK was identifiable by Western blot in ESO. lLK activity, measured by a filter assay in esophageal smooth muscle, increased significantly 30 and 60 seconds after ACh stimulation and was maximal at 60 seconds. The increased ILK activity at 60 seconds was inhibited by the ERK1/ERK2 MAP kinase inhibitor PD98059, but not by the p38 MAP kinase inhibitor SB203580, suggesting that ILK is activated by ERK1/ERK2, and not by p38 MAP kinase. In sapoinn-permeabilized ESO cells ILK antibody reduced (approximately 50%) ACh-induced contraction. The ILK-antibody in combination with the ERK1/ERK2 kinase inhibitor PD98059 caused the same reduction in contraction as PD98059 or the antibody alone, confimung that both ILK and ERK1/ERK2 may be in the same contractile pathway. ILKantibody inhibition was increased by the p38 MAP kinase inhibitor SB203580, indicating that ILK and the p38 MAP kinase may be in different contractile pathways. Conversely, an HSP27 antibody partially reduced (approximately 40%) ACh-induced contraction. HSP27 inhibition was not increased by the p38 MAP kinase inhibitor SB203580, but was increased by the ERK1/ERK2 MAP kinase inhibitor PD98059, suggesting that p38 MAP kinase and HSP27 are in the same pathway. Finally, antibodies against HSP27 and ILK, when used in combination, almost abolished ACh-induced contraction. We conclude that ACh-induced contraction of cat ESO is mediated through a dual pathway: one involving ERK1/ERK2 and ILK and the other involving p38 MAP kinase and HSP27. Supported by NIH RO1-DK28614 and C1HR MOP-13101.
Sl138 Is Ineffective Peristalsis Really Ineffective? Nam Q. Nguyen, Rachael Rigda, Marcus Tippett, Adreas J. Smout, Richard H Holloway Ineffective esophageal motility, defined as >30% pressure waves in the distal oesophagus <30 mmHg, has been suggested to be associated with impaired oesophageal clearance. However, this association has been based on either radiological or scintigraphic assessment of bolus clearance. Multiple intraluminal impedance (Mll) has been proposed as a nonradiological tool for studying esophageal volume clearance. Aims. The aim of this study was to assess the impact of peristaltic wave amplitude on oesophageal volume clearance using MI1. Methods. Concurrent perfusion manometry and MII was performed in 25 healthy asymptomatic volunteers (16M, 9F, 20-77 yr). Esophageal motility was measured at 4 sites 5-cm apart, starting 2cm above the lower esophageal sphincter (LES). MI1 was recorded at corresponding sites with electrodes incorporated into the manometric assembly. Ten, 5-ml liquid (saline) boluses were tested in each subject in the right-lateral position. Normal values for bolus presence time (BPT) at each site and total bolus transit time (TSTT) were derived from peristaltic sequences deemed to be manometrically normal (peristaltic propagation, wave amplitudes >35 mmHg). BPT and TBTT were then measured for all responses and the proportion of boluses cleared at each site as well as TBTT were correlated with wave amplitude. Results. The proportion of boluses cleared at each site was high and did not increase significantly above a threshold of 20 mmHg (Table). Peristaltic responses with at least all pressure waves > 30 mmHg were associated with normal BPT in 88% and normal TBTT in 98% of responses. When at least one wave was <30 mmHg normal BPT and TBTT were seen in 77% and 89% respectively. When the minimum amplitude was <15 mmHg, BPT and TBTT were normal in 67% and 80%. Conclusions. Esophageal clearance of a liquid bolus from the distal esophagus is effective at pressure wave amplitudes below 30 mmHg and in most regions as low as 11 mmHg. These data suggest that the current threshold for the definition of ineffective peristalsis is too high.
S1141
Spontaneous Tone and Cholinergic Contractions: Differences in Calcium Handling in Cat Lower Esophageal Sphincter Sling and Clasp Muscles
% Normal BPT and TB'I'T related to wave amplitude at Impedance segment Impedance sagmeat (cm above LES) '12 cm BPT 12 cm TBTT
0-10 mmHo
1t.20 mmHg
21.30 mmHg
31-40 rnmH9
4t.50 mmHg
> 50 mmHg
93%
93%
89%
96%
97%
77%*
80%
100%
87% 87%
97% 100% 81%
93% 83% 83%
90% 100% 100%
1 cm BPT 13% * 88% 89% Tom TB'l-r 25% * 100% 94% 2 cm BPT 50%" 78% ** 91% 2cm TBTT 70% 77% *~ 91% ' P
Ahmad Mninuddin, Leifa Neshatian, Nicholas E. Diamant Background: There are differences in length tension relationships and cholinerglc sensitivity of sling and clasp muscles in the cat lower esophageal sphincter (LES). Hypothesis: There are differences in the sources of Ca2* utilized for contraction in sling and clasp muscles. Methods: Studies were performed on muscle strips from sling and clasp regions of the LES in the presence of tetrodotoxin. Results: 1)Tone: role of extracellular (EC) Ca 2+. Clasp muscle developed more spontaneous tone than sling muscle (p<.05). In Ca2§ free Krebs or in the presence of the L-type Ca 2* channel blocker infedipine, clasp exhibited a greater decrease in tone (p<.05). 2)Tone: role of Ca 2§ from internal stores. The sarcoplasmic reticuhim (SR) Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), caused greater increase in tone in sling than in clasp muscle(p<.02). Stimulation of ryanodine sensitive stores with caffeine inhibited tone in both muscles. Inhibition of ryanodine receptors with ryanodine caused a similar increase in tone in sling and clasp. The SR IP3 receptor blocker 2-APB abolished tone in clasp with no inhibition in sling. 3)Cbolinergic contractions: role of EC Cat+. Acetylcholineinduced contractions (CC) were of greater amplitude in sling than in clasp muscle(p<.05). In Ca2+ free Krebs, CC were similarly inhibited in a time and challenge dependent manner in both muscles. Nifedipine abolished CC in the clasp but only slightly inhibited the CC in the sling. 4)Cholinerglc contractions: role of Ca2+ from internal stores. CPA, caffeine and ryanodine caused a similar degree of inhibition of CC in both sling and clasp muscle. 2APB completely abolished CC in both muscles. Conclusions: In sling and clasp muscle, intracelhilar (IC) and EC Ca 2+ sources are utilized to different degrees in the generation of
100%
95% 98%
97% 98%
51139 Rho Kinase and Cat Lower Esophageal Sphincter (LES) Tone Weibiao Cao, Karen M. Harnett, Ling Cheng, Jose Behar, Piero Bianeani We have previously shown that PGF2a participates in maintenance of LES tone and that the initial PGF2a-induced contraction depends on activation of Gq and G,3. It has recently been shown (Hersh et al . Gastroenterology 2002, 122 Suppl: A-256) that the pathways mediating the initial phase of contraction in response to exogenously administered agonists
A-161
AGA
Abstracts
may be different from those mediating sustained contraction, which may depend on a Rhokinase pathway. We therefore tested the proposition that sustained LES tone, which depends on elevated levels of endogenous PGF2a may be maintained through a Rho-kinase dependent pathway. In LES circular muscle strips the Rho-kinase inhibitor Y27632 dose-dependently decreased tone and abolished it at 10 ~ M, suggesting that LES tone is Rho-kinase- dependent. The PGF2a antagonist AL-8810 dose-dependently reduced tone. In strips treated with indomethacin to abolish production of endogenous PGF2a, exogenously administered PGF2a maintained a sustained contraction lasting more than 30 rain. The sustained, but not the initial contraction in response to PGF2a was abolished by Y27632. LES circular smooth muscle cells, isolated by enzymatic digestion were used to exanaine the intracelhilar contractile pathways mediating Rho-kinase- PGF2a-dependent sustained contraction. In cells PGF2a caused a relatively rapid contraction that achieved maximum shortening in approximately 30 seconds and persisted in excess of 20 minutes. After treatment with the Rho-kinase inhibitor Y27632 cells achieved the same maximum initial shortening as untreated ceils, but did not remain contracted, and returned to their unstimulated length within approximately 10 minutes. The inhibition of sustained contraction by Y27632 in cells mimics the inhibition of tone and of sustained PGF2a-induced contraction in strips In saponin-permeabilized cells, PGF2a-induced shortening was the same as in intact ceils. In these permeable cells G~3 and RhoA antibodies reduced and the selective RhoA inhibitor C3 (exoenzyme of Clostridium Botulinum) abolished sustained contraction induced by PGF2a, confirming that the contraction is mediated by RhoA-induced activation of Rhokinase. The data suggest a role of RhoA- Rho-Kinase in maintenance of PGF2a induced sustained contraction and LES tone. Supported by NIH ROI-DK-28614.
Not Just a Failure to Relax: Timing and Magnitude of Lower Esophageal Sphincter After-Contractions Also Contribute to Esophageal Outflow Obstruction in Achalasia Samer Gawrieh, Michael C. Jean, Benson T. Massey BACKGROUND: Impaired esophageal emptying is a cardinal finding in achalasia and is traditionally felt to result from failed lower esophageal sphincter (LES) relaxation and absent esophageal body peristalsis, little attention has been given to the presence of deglutitive after-conrtactions (ACs) in achalasia and their possible effect on esophageal emptying. METHODS: Manomet~ac recordings from 68 achalasia patients (36 F, mean 47 115-85] yr) referred to our clinical manometry laboratory were analyzed. Studies were performed with the subjects lasting and supine, using a sleeve device to record from the LES. The following parameters were scored for three 5ml water swallows: Initial LES pressure (LESP); time (from swallow onset) and magnitude of peak esophageal body pressure (EBP) wave (located 2 cm above the top of the LES); incidence, time, and magnitude of LES deglutitive AC; maximum value by which EBP exceeded LESP during any time after the swallow; basal EflP between swallows. In addition, whether EBP waveforms were isobaric or non-isobaric was recorded. Values were compared to those in 16 subjects (8F, mean 47 [14-881 yr) with normal peristalsis and LES relaxation. RESULTS: The incidence of LES AC was similar in achalasia and controls (84% vs. 92%). As expected, basal LESP was higher (31 vs. 22 mmHg, p<.001) and peak EBP was lower (34 vs. 93 mmHg, p<.001) in achalasia. In achalasia, time of peak EPB was earlier (4.5 vs. 8.2 s, p<.O01) and peak EBP generally exceeded basal LESP (p<.001). However, LES ACs also occurred earlier in achalasia (5.8 vs. 9.8 s, p<.001) and typically exceeded peak EBP (55 mmHg vs. 34 mmHg, p<.O01). As a result, the EBPLESP differential did not favor emptying in achalasia, whereas it did in controls (-4 vs. 13 mmHg, p<.001). The worse this differential, the higher the basal EBP was in achalasia patients (p= .008). Swallows showing non-isobaric waveforms tended to have a favorable pressure differential compared to those that were isobaric (3 vs. -4 mmHg, p = .026). In a multiple regression analysis, factors sigraficantly determining the pressure differential were: peak EBP (positively); basal LESP, time of LES AC and magnitude of LES AC (negatively). Higher peak EBP were offset by their association with higher LES AC (p<.001). CONCLUSIONS: In achalasia, esophageal outflow obstruction is not simply a matter of the LES not relaxing. Timing and magnitude of LES ACs also contribute to this obstruction. Supported by NIH PO1 03191-03.
Sl140 Integrin Linked Kinase (ILK) in ACh-lnduced Contraction of Cat Esophageal Circular Smooth Muscle (ESO) Weibiao Cao, Ling Chang, Karen M. Hamett, .ling T. Dang, Jose Behar, Michael P. Walsh, Piero Biancani We have previously shown that ACh-induced contraction of cat ESO is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid, activation of the Cat§ -insensitive protein kinase C-~ (PKCE) and activation of two distinct MAP kinase pathways: an ERK1/ERK2 MAP kinase-dependent pathway and an HSP27-1inked p38 kinasedependent pathway. The sequence of events occurring between activation of MAP kinases and muscle contraction, however, has not been elucidated. A possible candidate intermediate protein in Ca2+-independent PKCt-mediated contraction may be ILK, a kinase associated with myofilaments and responsible for Ca2§ -independent phnsphorylation of smooth muscle myosin. We therefore examined the role of ILK in ACh-induced contraction of cat ESO. ILK was identifiable by Western blot in ESO. lLK activity, measured by a filter assay in esophageal smooth muscle, increased significantly 30 and 60 seconds after ACh stimulation and was maximal at 60 seconds. The increased ILK activity at 60 seconds was inhibited by the ERK1/ERK2 MAP kinase inhibitor PD98059, but not by the p38 MAP kinase inhibitor SB203580, suggesting that ILK is activated by ERK1/ERK2, and not by p38 MAP kinase. In sapoinn-permeabilized ESO cells ILK antibody reduced (approximately 50%) ACh-induced contraction. The ILK-antibody in combination with the ERK1/ERK2 kinase inhibitor PD98059 caused the same reduction in contraction as PD98059 or the antibody alone, confimung that both ILK and ERK1/ERK2 may be in the same contractile pathway. ILKantibody inhibition was increased by the p38 MAP kinase inhibitor SB203580, indicating that ILK and the p38 MAP kinase may be in different contractile pathways. Conversely, an HSP27 antibody partially reduced (approximately 40%) ACh-induced contraction. HSP27 inhibition was not increased by the p38 MAP kinase inhibitor SB203580, but was increased by the ERK1/ERK2 MAP kinase inhibitor PD98059, suggesting that p38 MAP kinase and HSP27 are in the same pathway. Finally, antibodies against HSP27 and ILK, when used in combination, almost abolished ACh-induced contraction. We conclude that ACh-induced contraction of cat ESO is mediated through a dual pathway: one involving ERK1/ERK2 and ILK and the other involving p38 MAP kinase and HSP27. Supported by NIH RO1-DK28614 and C1HR MOP-13101.
Sl138 Is Ineffective Peristalsis Really Ineffective? Nam Q. Nguyen, Rachael Rigda, Marcus Tippett, Adreas J. Smout, Richard H Holloway Ineffective esophageal motility, defined as >30% pressure waves in the distal oesophagus <30 mmHg, has been suggested to be associated with impaired oesophageal clearance. However, this association has been based on either radiological or scintigraphic assessment of bolus clearance. Multiple intraluminal impedance (Mll) has been proposed as a nonradiological tool for studying esophageal volume clearance. Aims. The aim of this study was to assess the impact of peristaltic wave amplitude on oesophageal volume clearance using MI1. Methods. Concurrent perfusion manometry and MII was performed in 25 healthy asymptomatic volunteers (16M, 9F, 20-77 yr). Esophageal motility was measured at 4 sites 5-cm apart, starting 2cm above the lower esophageal sphincter (LES). MI1 was recorded at corresponding sites with electrodes incorporated into the manometric assembly. Ten, 5-ml liquid (saline) boluses were tested in each subject in the right-lateral position. Normal values for bolus presence time (BPT) at each site and total bolus transit time (TSTT) were derived from peristaltic sequences deemed to be manometrically normal (peristaltic propagation, wave amplitudes >35 mmHg). BPT and TBTT were then measured for all responses and the proportion of boluses cleared at each site as well as TBTT were correlated with wave amplitude. Results. The proportion of boluses cleared at each site was high and did not increase significantly above a threshold of 20 mmHg (Table). Peristaltic responses with at least all pressure waves > 30 mmHg were associated with normal BPT in 88% and normal TBTT in 98% of responses. When at least one wave was <30 mmHg normal BPT and TBTT were seen in 77% and 89% respectively. When the minimum amplitude was <15 mmHg, BPT and TBTT were normal in 67% and 80%. Conclusions. Esophageal clearance of a liquid bolus from the distal esophagus is effective at pressure wave amplitudes below 30 mmHg and in most regions as low as 11 mmHg. These data suggest that the current threshold for the definition of ineffective peristalsis is too high.
S1141
Spontaneous Tone and Cholinergic Contractions: Differences in Calcium Handling in Cat Lower Esophageal Sphincter Sling and Clasp Muscles
% Normal BPT and TB'I'T related to wave amplitude at Impedance segment Impedance sagmeat (cm above LES) '12 cm BPT 12 cm TBTT
0-10 mmHo
1t.20 mmHg
21.30 mmHg
31-40 rnmH9
4t.50 mmHg
> 50 mmHg
93%
93%
89%
96%
97%
77%*
80%
100%
87% 87%
97% 100% 81%
93% 83% 83%
90% 100% 100%
1 cm BPT 13% * 88% 89% Tom TB'l-r 25% * 100% 94% 2 cm BPT 50%" 78% ** 91% 2cm TBTT 70% 77% *~ 91% ' P
Ahmad Mninuddin, Leifa Neshatian, Nicholas E. Diamant Background: There are differences in length tension relationships and cholinerglc sensitivity of sling and clasp muscles in the cat lower esophageal sphincter (LES). Hypothesis: There are differences in the sources of Ca2* utilized for contraction in sling and clasp muscles. Methods: Studies were performed on muscle strips from sling and clasp regions of the LES in the presence of tetrodotoxin. Results: 1)Tone: role of extracellular (EC) Ca 2+. Clasp muscle developed more spontaneous tone than sling muscle (p<.05). In Ca2§ free Krebs or in the presence of the L-type Ca 2* channel blocker infedipine, clasp exhibited a greater decrease in tone (p<.05). 2)Tone: role of Ca 2§ from internal stores. The sarcoplasmic reticuhim (SR) Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), caused greater increase in tone in sling than in clasp muscle(p<.02). Stimulation of ryanodine sensitive stores with caffeine inhibited tone in both muscles. Inhibition of ryanodine receptors with ryanodine caused a similar increase in tone in sling and clasp. The SR IP3 receptor blocker 2-APB abolished tone in clasp with no inhibition in sling. 3)Cbolinergic contractions: role of EC Cat+. Acetylcholineinduced contractions (CC) were of greater amplitude in sling than in clasp muscle(p<.05). In Ca2+ free Krebs, CC were similarly inhibited in a time and challenge dependent manner in both muscles. Nifedipine abolished CC in the clasp but only slightly inhibited the CC in the sling. 4)Cholinerglc contractions: role of Ca2+ from internal stores. CPA, caffeine and ryanodine caused a similar degree of inhibition of CC in both sling and clasp muscle. 2APB completely abolished CC in both muscles. Conclusions: In sling and clasp muscle, intracelhilar (IC) and EC Ca 2+ sources are utilized to different degrees in the generation of
100%
95% 98%
97% 98%
51139 Rho Kinase and Cat Lower Esophageal Sphincter (LES) Tone Weibiao Cao, Karen M. Harnett, Ling Cheng, Jose Behar, Piero Bianeani We have previously shown that PGF2a participates in maintenance of LES tone and that the initial PGF2a-induced contraction depends on activation of Gq and G,3. It has recently been shown (Hersh et al . Gastroenterology 2002, 122 Suppl: A-256) that the pathways mediating the initial phase of contraction in response to exogenously administered agonists
A-161
AGA
Abstracts