Reviewer's report 
 Effects of a Cholesterol-Enriched Diet on Intestinal Smooth Muscle Contraction: Inhibition of Title: 
Muscarinic Receptors and their Disinhibition by the 5-HT4 Agonist - Tegaserod 
 1  12 October 2005 Version: Date: 
 David Schneider Reviewer: 
Reviewer's report: 
General 
This is a new and well conducted study that demonstrates enhanced cholinergic contraction of the 
jejunum of ground squirrels on a high cholesterol diet. The result is somewhat surprising in that it 
has been previously shown that the muscarinic contraction of gallbladder smooth muscle is reduced 
in this model. The present study shows that enhanced jejunal contraction is due to loss of a 
competing muscarinic-induced nerve-mediated inhibition of jejunal smooth muscle. Some of the evidence presented suggests that the mechanism is dependent upon development of increased 
biliary cholesterol, at least within the hepatic duct. The results also uncover a novel effect of 
muscarinic stimulation in producing nerve-mediated smooth muscle inhibition in the jejunum, an 
effect not seen in the ileum. It is unfortunate that the effects of tegaserod on the distal small intestine 
were not also studied, as the enhanced peristalsis expected from tegaserod might have also 
affected the ileal exposure to luminal bile and increased cholesterol. The present findings, 
nonetheless, are new and informative. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. Statements need to clarify whether or not any change in baseline tone or spontaneous activity 
was observed in the presence of TTX, hexamethonium and scopolamine and, if so, how that was 
handled in data interpretation. 
2. This article does not establish whether chronic (>28d) exposure was necessary to observe the 
effects of high cholesterol diet. Is there another reference that provides similar information for acute 
exposure in ground squirrels? If not, please indicate that the necessary time to develop enhanced 
jejunal contraction is not known and could have been an acute effect. 
3. This study did not distinguish between muscarinic receptor transduction mechanisms (i.e., 
inhibitory versus excitatory) (See first line of page 13). Strictly speaking, this study demonstrates a unique jejunal smooth muscle inhibitory mechanism that is dependent upon stimulation of an unknown type of muscarinic receptor and the activation of VGSCs. Please correct any wording that 
implies the observed mechanism necessarily results from stimulation of muscarinic receptors that 
are directly coupled to inhibitory transduction mechanisms (e.g., M2 receptors). 
4. With regard to the last sentence in middle paragraph on page 14: Pre-treatments with tegaserod 
are not equivalent to placing tegaserod in the tissue bath as in the referenced experiments (44-48, 
51). Tegaserod certainly washes out in the tissue bath unless it is re-added. Tegaserod effects in your experiment must be prior to in vitro testing. Please correct this statement and reconsider tegaserod conclusions. 
5. The first sentence of the last paragraph on page 14 is potentialy misleading. High cholesterol diet 
appears to have disinhibited muscarinic contraction of jejunal smooth muscle similar to the effects of 
TTX. Since tegaserod reduced hepatic duct cholesterol, however, the effect of tegaserod may have 
been normalization of cholesterol exposure despite a continued high cholesterol diet. Note: Even though you found no statistical effect of tegaserod on gallbladder cholesterol in animals receiving high cholesterol diet, you have not tested whether or not gallbladder cholesterol remained elevated 
above baseline – i.e., above gallbladder cholesterol in non-treated animals on a low cholesterol diet. 
It appears unlikely that it remains significantly increased in tegaserod treated animals on high 
cholesterol diets. Therefore, instead of a reversal of the disinhibition, it seems as likely that tegaserod may have prevented the increased cholesterol necessary to produce the effect. Please 
consider these comments and reconsider tegaserod conclusions. 
6. The authors need to discuss the disparate effects of high cholesterol diet on gallbladder versus 
jejunal versus ileal muscarinic contraction. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
7. Methods; Experimental Animals: Considering these were wild animals used for experimentation 
within a month of capture, can you state anything about the uniform health of these animals, 
especially as relates to internal parasitism? Were animals given an anthelmintic and, if so, what? 
8. Methods; Experimental Animals, Page 6: “Animals randomly divided into four groups (19 in 
each)…”; so, this accounts for 76 of the 96 animals used. Please explain how the other 20 were 
used or make corrections as necessary. The very next paragraph does not explain which animals 
were used for the determination of hepatic and gallbladder bile cholesterol concentration? Please 
clarify. 
9. Cholesterol Concentrations in Hepatic and Gallbladder Bile, page 6: Please define “acute” in 
“acute terminal experiments in vivo…” Were these animals in addition to those used in the 
contractility experiments? Please clarify. 
10. First line of page 7: remove extraneous “(“. 
11. First sentence under Intestinal Contractility in vitro, page 7: (a) Please clarify. Were experiments 
started after 4 weeks on diet by testing 2 animals per diet group daily? (b) Please clarify how 
preparations from each animal were used. Was it one jejunum/one ileum for carbachol alone, one 
jejunum/one ileum for carbachol in the presence of TTX, one jejunum for carbachol in the presence 
of hexamethonium, leaving one jejunum for carbachol in the presence of scopolamine? In the 
results (page 10), however, it is stated that ileal contraction was not modified by hexamethonium. In 
which animals were the effects of hexamethonium studied on ileal segments? 
12. Intestinal contractility in vitro, page 7: (a) Please state temperature of tissue bath system. (b) 
Please clarify if bethanechol was added cumulatively or non-cumulatively? If the latter, in what order 
were concentrations added? (c) Please state how long antagonists were allowed to pre-incubate 
before constructing concentration-response curves? 
13. Intestinal contractility in vitro, page 7: 0.1 µM TTX is pretty minimal; 0.3 µM is generally needed 
in guinea pigs and at least 1 µM in mice and rats to reliably block nerve-mediated effects. Is there 
any reference that documents this concentration is sufficient to block nerve-mediated activity (MMC, 
peristalsis, or electric-field induced)? Nonetheless, you observed an effect that might best be 
qualified as highly sensitive to block by TTX. 
14. Reference [9] (top of page 8) is a study of the migrating myoelectric complex and does not 
involve stress measurements. Please provide appropriate reference. 
15. Page 8: Please fix subject verb agreement in second line: “…, the tissue preparation were…”. 
Please consider revising second sentence of Data Analysis. 
16. Data Analysis, page 8: Consider revising second sentence. Also, it should not be a mystery as to 
which statistical tests were used for each experiment. If not listed here, then it should be made clear 
in the Results or in the figure legends. [In my opinion, the cholesterol data should be tested with a 
two-way analysis of variance. The same could be done for predicted maximum effects for 
concentration-response curves. Addressing this additional comment is not essential as I don’t 
believe reanalysis would result in a change in the statistical differences found and not found. Power 
may have been a problem for the cholesterol measurements in the gallbladder.] 
17. Cholinergic Contractions of Jejunal Longitudinal Smooth Muscle, page 9: (a) Correct first 
sentence structure “…carbachol with increasing in tonic contractions…” and delete “optimal” as there is no indication as to why it is optimal; perhaps supramaximal. (b) Correct first sentence, second paragraph “…Newtons)/m2…” 
18. Effect of Action Potential Blockade on Carbachol-Evoked Contractions, page 9: (a) “Following 
TTX exposure …” is not correct. It was “In the presence of TTX …” (b) Second paragraph page 10: First sentence is awkward. Is the point that the TTX-induced enhancement of cholinergic jejunal 
contractions was not altered by pre-treatment with tegaserod in the low-cholesterol diet group? (c) 
Please make clear whether or not the effects of hexamethonium and scopolamine were tested on 
animals treated with tegaserod in the jejunum and ileum. 
19. Interactions…, page 11: Please correct text since you DO show the hexamethonium data in high 
cholesterol diet animals (Figure 3b). 
20. References are expected for line one of page 12: “…influences the affinity, binding capacity and 
signal transduction.” Especially references related to such effects on muscarinic receptors. 
21. Bottom of page 13: (a) Reference 41 only shows increased cholesterol content in gallbladder 
smooth muscle, which has reduced (not enhanced) contractions. It may be too much to assume that 
jejunal smooth muscle or jejunal myenteric neuron membranes also have increased cholesterol 
content. Direct exposure of myenteric motor neurons to lumen and absorbed substances does not 
seem likely. Note also that exposure to plasma cholesterol (previously shown to be elevated in these 
animals) would likely be similar for myenteric neurons of jejunum and ileum. (b) High cholesterol diet 
reduced an opposing inhibitory mechanism to the smooth muscle that is mediated by activation of a 
muscarinic receptor and involves opening of TTX-sensitive VGSCs. Might the effects of high cholesterol diet on this inhibitory mechanism be due to loss of receptors, loss of receptor transduction mechanism, and/or loss of inhibitory mechanism? 
22. Please clarify last sentence of page 13 that continues on to page 14. 
23. Figures (a) X-axis in all figures should be “log[Carbachol](M)” and not “[Carbachol].M”. (b) 
Correct supra-scripted end-bracket in Y-axis labels in all figures. (c) Figure 2: correct the spelling of 
tegaserod. (d) For future reference and potential comparison, you must present more detail of the data on ileal contractions. This might be easiest to achieve by inclusion of the concentration-response relationship figure. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
24. On page 2 within the Background of the Abstract: it is probably sufficient to use “5-HT4 receptor” 
instead of “5-HT4 serotonin receptor”. The latter is redundant and not used anywhere else in the 
manuscript. 
25. For clarity throughout the manuscript, consider consistent referral to the diets as low versus high 
cholesterol with definition given in the Methods. Within the abstract, for instance, the diets are 
referred to as low, 1% or enriched, and in the Results as “normal”. 
26. Eighth line page 5: It is not clear why “drugs used to treat this condition should have minimal 
impact on gallbladder contractility.” Would a drug that increased biliary emptying not be potentially 
helpful in decreasing the likelihood of stone formation? In either case, your data show a significant 
reduction in hepatic duct cholesterol with chronic tegaserod. Is that to be viewed as unfavorable? 
27. Regarding the normalization method: Although only a minor concern, are the authors certain that 
normalization using this formula remains appropriate after high cholesterol diet? In other words, is it 
known that the relationship of longitudinal smooth muscle area to whole preparation weight and length remains unchanged with diet? For instance, some diets reduce or increase intestinal length. While normalization of data is frequently helpful, do the basic relationships hold up if the data is not 
normalized in this way? 
28. In my opinion, there is no need to repeatedly state “significantly… (P<0.05)” throughout the 
manuscript when you’ve already defined significance as P<0.05 in the Methods. The exception 
would be for figure legends, which should be able to be read alone. 
29. It might be worthwhile noting in the discussion that these animals are expected to be 
hypercholesterolemic such that a difference between jejunum and ileum may have been luminal 
exposure to increased cholesterol or bile salts but probably not in exposure to plasma cholesterol. 
30. Figures 1 and 2 show the same data, but in a different combination. In my opinion, Figure 1 is not 
necessary. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
Reviewer's report 
 Effects of a Cholesterol-Enriched Diet on Intestinal Smooth Muscle Contraction: Inhibition of Title: 
Muscarinic Receptors and their Disinhibition by the 5-HT4 Agonist - Tegaserod 
 1  12 October 2005 Version: Date: 
 David Schneider Reviewer: 
Reviewer's report: 
General 
This is a new and well conducted study that demonstrates enhanced cholinergic contraction of the 
jejunum of ground squirrels on a high cholesterol diet. The result is somewhat surprising in that it 
has been previously shown that the muscarinic contraction of gallbladder smooth muscle is reduced 
in this model. The present study shows that enhanced jejunal contraction is due to loss of a 
competing muscarinic-induced nerve-mediated inhibition of jejunal smooth muscle. Some of the evidence presented suggests that the mechanism is dependent upon development of increased 
biliary cholesterol, at least within the hepatic duct. The results also uncover a novel effect of 
muscarinic stimulation in producing nerve-mediated smooth muscle inhibition in the jejunum, an 
effect not seen in the ileum. It is unfortunate that the effects of tegaserod on the distal small intestine 
were not also studied, as the enhanced peristalsis expected from tegaserod might have also 
affected the ileal exposure to luminal bile and increased cholesterol. The present findings, 
nonetheless, are new and informative. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. Statements need to clarify whether or not any change in baseline tone or spontaneous activity 
was observed in the presence of TTX, hexamethonium and scopolamine and, if so, how that was 
handled in data interpretation. 
2. This article does not establish whether chronic (>28d) exposure was necessary to observe the 
effects of high cholesterol diet. Is there another reference that provides similar information for acute 
exposure in ground squirrels? If not, please indicate that the necessary time to develop enhanced 
jejunal contraction is not known and could have been an acute effect. 
3. This study did not distinguish between muscarinic receptor transduction mechanisms (i.e., 
inhibitory versus excitatory) (See first line of page 13). Strictly speaking, this study demonstrates a unique jejunal smooth muscle inhibitory mechanism that is dependent upon stimulation of an unknown type of muscarinic receptor and the activation of VGSCs. Please correct any wording that 
implies the observed mechanism necessarily results from stimulation of muscarinic receptors that 
are directly coupled to inhibitory transduction mechanisms (e.g., M2 receptors). 
4. With regard to the last sentence in middle paragraph on page 14: Pre-treatments with tegaserod 
are not equivalent to placing tegaserod in the tissue bath as in the referenced experiments (44-48, 
51). Tegaserod certainly washes out in the tissue bath unless it is re-added. Tegaserod effects in your experiment must be prior to in vitro testing. Please correct this statement and reconsider tegaserod conclusions. 
5. The first sentence of the last paragraph on page 14 is potentialy misleading. High cholesterol diet 
appears to have disinhibited muscarinic contraction of jejunal smooth muscle similar to the effects of 
TTX. Since tegaserod reduced hepatic duct cholesterol, however, the effect of tegaserod may have 
been normalization of cholesterol exposure despite a continued high cholesterol diet. Note: Even though you found no statistical effect of tegaserod on gallbladder cholesterol in animals receiving high cholesterol diet, you have not tested whether or not gallbladder cholesterol remained elevated 
above baseline – i.e., above gallbladder cholesterol in non-treated animals on a low cholesterol diet. 
It appears unlikely that it remains significantly increased in tegaserod treated animals on high 
cholesterol diets. Therefore, instead of a reversal of the disinhibition, it seems as likely that tegaserod may have prevented the increased cholesterol necessary to produce the effect. Please 
consider these comments and reconsider tegaserod conclusions. 
6. The authors need to discuss the disparate effects of high cholesterol diet on gallbladder versus 
jejunal versus ileal muscarinic contraction. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
7. Methods; Experimental Animals: Considering these were wild animals used for experimentation 
within a month of capture, can you state anything about the uniform health of these animals, 
especially as relates to internal parasitism? Were animals given an anthelmintic and, if so, what? 
8. Methods; Experimental Animals, Page 6: “Animals randomly divided into four groups (19 in 
each)…”; so, this accounts for 76 of the 96 animals used. Please explain how the other 20 were 
used or make corrections as necessary. The very next paragraph does not explain which animals 
were used for the determination of hepatic and gallbladder bile cholesterol concentration? Please 
clarify. 
9. Cholesterol Concentrations in Hepatic and Gallbladder Bile, page 6: Please define “acute” in 
“acute terminal experiments in vivo…” Were these animals in addition to those used in the 
contractility experiments? Please clarify. 
10. First line of page 7: remove extraneous “(“. 
11. First sentence under Intestinal Contractility in vitro, page 7: (a) Please clarify. Were experiments 
started after 4 weeks on diet by testing 2 animals per diet group daily? (b) Please clarify how 
preparations from each animal were used. Was it one jejunum/one ileum for carbachol alone, one 
jejunum/one ileum for carbachol in the presence of TTX, one jejunum for carbachol in the presence 
of hexamethonium, leaving one jejunum for carbachol in the presence of scopolamine? In the 
results (page 10), however, it is stated that ileal contraction was not modified by hexamethonium. In 
which animals were the effects of hexamethonium studied on ileal segments? 
12. Intestinal contractility in vitro, page 7: (a) Please state temperature of tissue bath system. (b) 
Please clarify if bethanechol was added cumulatively or non-cumulatively? If the latter, in what order 
were concentrations added? (c) Please state how long antagonists were allowed to pre-incubate 
before constructing concentration-response curves? 
13. Intestinal contractility in vitro, page 7: 0.1 µM TTX is pretty minimal; 0.3 µM is generally needed 
in guinea pigs and at least 1 µM in mice and rats to reliably block nerve-mediated effects. Is there 
any reference that documents this concentration is sufficient to block nerve-mediated activity (MMC, 
peristalsis, or electric-field induced)? Nonetheless, you observed an effect that might best be 
qualified as highly sensitive to block by TTX. 
14. Reference [9] (top of page 8) is a study of the migrating myoelectric complex and does not 
involve stress measurements. Please provide appropriate reference. 
15. Page 8: Please fix subject verb agreement in second line: “…, the tissue preparation were…”. 
Please consider revising second sentence of Data Analysis. 
16. Data Analysis, page 8: Consider revising second sentence. Also, it should not be a mystery as to 
which statistical tests were used for each experiment. If not listed here, then it should be made clear 
in the Results or in the figure legends. [In my opinion, the cholesterol data should be tested with a 
two-way analysis of variance. The same could be done for predicted maximum effects for 
concentration-response curves. Addressing this additional comment is not essential as I don’t 
believe reanalysis would result in a change in the statistical differences found and not found. Power 
may have been a problem for the cholesterol measurements in the gallbladder.] 
17. Cholinergic Contractions of Jejunal Longitudinal Smooth Muscle, page 9: (a) Correct first 
sentence structure “…carbachol with increasing in tonic contractions…” and delete “optimal” as there is no indication as to why it is optimal; perhaps supramaximal. (b) Correct first sentence, second paragraph “…Newtons)/m2…” 
18. Effect of Action Potential Blockade on Carbachol-Evoked Contractions, page 9: (a) “Following 
TTX exposure …” is not correct. It was “In the presence of TTX …” (b) Second paragraph page 10: First sentence is awkward. Is the point that the TTX-induced enhancement of cholinergic jejunal 
contractions was not altered by pre-treatment with tegaserod in the low-cholesterol diet group? (c) 
Please make clear whether or not the effects of hexamethonium and scopolamine were tested on 
animals treated with tegaserod in the jejunum and ileum. 
19. Interactions…, page 11: Please correct text since you DO show the hexamethonium data in high 
cholesterol diet animals (Figure 3b). 
20. References are expected for line one of page 12: “…influences the affinity, binding capacity and 
signal transduction.” Especially references related to such effects on muscarinic receptors. 
21. Bottom of page 13: (a) Reference 41 only shows increased cholesterol content in gallbladder 
smooth muscle, which has reduced (not enhanced) contractions. It may be too much to assume that 
jejunal smooth muscle or jejunal myenteric neuron membranes also have increased cholesterol 
content. Direct exposure of myenteric motor neurons to lumen and absorbed substances does not 
seem likely. Note also that exposure to plasma cholesterol (previously shown to be elevated in these 
animals) would likely be similar for myenteric neurons of jejunum and ileum. (b) High cholesterol diet 
reduced an opposing inhibitory mechanism to the smooth muscle that is mediated by activation of a 
muscarinic receptor and involves opening of TTX-sensitive VGSCs. Might the effects of high cholesterol diet on this inhibitory mechanism be due to loss of receptors, loss of receptor transduction mechanism, and/or loss of inhibitory mechanism? 
22. Please clarify last sentence of page 13 that continues on to page 14. 
23. Figures (a) X-axis in all figures should be “log[Carbachol](M)” and not “[Carbachol].M”. (b) 
Correct supra-scripted end-bracket in Y-axis labels in all figures. (c) Figure 2: correct the spelling of 
tegaserod. (d) For future reference and potential comparison, you must present more detail of the data on ileal contractions. This might be easiest to achieve by inclusion of the concentration-response relationship figure. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
24. On page 2 within the Background of the Abstract: it is probably sufficient to use “5-HT4 receptor” 
instead of “5-HT4 serotonin receptor”. The latter is redundant and not used anywhere else in the 
manuscript. 
25. For clarity throughout the manuscript, consider consistent referral to the diets as low versus high 
cholesterol with definition given in the Methods. Within the abstract, for instance, the diets are 
referred to as low, 1% or enriched, and in the Results as “normal”. 
26. Eighth line page 5: It is not clear why “drugs used to treat this condition should have minimal 
impact on gallbladder contractility.” Would a drug that increased biliary emptying not be potentially 
helpful in decreasing the likelihood of stone formation? In either case, your data show a significant 
reduction in hepatic duct cholesterol with chronic tegaserod. Is that to be viewed as unfavorable? 
27. Regarding the normalization method: Although only a minor concern, are the authors certain that 
normalization using this formula remains appropriate after high cholesterol diet? In other words, is it 
known that the relationship of longitudinal smooth muscle area to whole preparation weight and length remains unchanged with diet? For instance, some diets reduce or increase intestinal length. While normalization of data is frequently helpful, do the basic relationships hold up if the data is not 
normalized in this way? 
28. In my opinion, there is no need to repeatedly state “significantly… (P<0.05)” throughout the 
manuscript when you’ve already defined significance as P<0.05 in the Methods. The exception 
would be for figure legends, which should be able to be read alone. 
29. It might be worthwhile noting in the discussion that these animals are expected to be 
hypercholesterolemic such that a difference between jejunum and ileum may have been luminal 
exposure to increased cholesterol or bile salts but probably not in exposure to plasma cholesterol. 
30. Figures 1 and 2 show the same data, but in a different combination. In my opinion, Figure 1 is not 
necessary. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
Please review this paper.
user
Reviewer's report 
 Effects of a Cholesterol-Enriched Diet on Intestinal Smooth Muscle Contraction: Inhibition of Title: 
Muscarinic Receptors and their Disinhibition by the 5-HT4 Agonist - Tegaserod 
 1  12 October 2005 Version: Date: 
 David Schneider Reviewer: 
Reviewer's report: 
General 
This is a new and well conducted study that demonstrates enhanced cholinergic contraction of the 
jejunum of ground squirrels on a high cholesterol diet. The result is somewhat surprising in that it 
has been previously shown that the muscarinic contraction of gallbladder smooth muscle is reduced 
in this model. The present study shows that enhanced jejunal contraction is due to loss of a 
competing muscarinic-induced nerve-mediated inhibition of jejunal smooth muscle. Some of the evidence presented suggests that the mechanism is dependent upon development of increased 
biliary cholesterol, at least within the hepatic duct. The results also uncover a novel effect of 
muscarinic stimulation in producing nerve-mediated smooth muscle inhibition in the jejunum, an 
effect not seen in the ileum. It is unfortunate that the effects of tegaserod on the distal small intestine 
were not also studied, as the enhanced peristalsis expected from tegaserod might have also 
affected the ileal exposure to luminal bile and increased cholesterol. The present findings, 
nonetheless, are new and informative. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. Statements need to clarify whether or not any change in baseline tone or spontaneous activity 
was observed in the presence of TTX, hexamethonium and scopolamine and, if so, how that was 
handled in data interpretation. 
2. This article does not establish whether chronic (>28d) exposure was necessary to observe the 
effects of high cholesterol diet. Is there another reference that provides similar information for acute 
exposure in ground squirrels? If not, please indicate that the necessary time to develop enhanced 
jejunal contraction is not known and could have been an acute effect. 
3. This study did not distinguish between muscarinic receptor transduction mechanisms (i.e., 
inhibitory versus excitatory) (See first line of page 13). Strictly speaking, this study demonstrates a unique jejunal smooth muscle inhibitory mechanism that is dependent upon stimulation of an unknown type of muscarinic receptor and the activation of VGSCs. Please correct any wording that 
implies the observed mechanism necessarily results from stimulation of muscarinic receptors that 
are directly coupled to inhibitory transduction mechanisms (e.g., M2 receptors). 
4. With regard to the last sentence in middle paragraph on page 14: Pre-treatments with tegaserod 
are not equivalent to placing tegaserod in the tissue bath as in the referenced experiments (44-48, 
51). Tegaserod certainly washes out in the tissue bath unless it is re-added. Tegaserod effects in your experiment must be prior to in vitro testing. Please correct this statement and reconsider tegaserod conclusions. 
5. The first sentence of the last paragraph on page 14 is potentialy misleading. High cholesterol diet 
appears to have disinhibited muscarinic contraction of jejunal smooth muscle similar to the effects of 
TTX. Since tegaserod reduced hepatic duct cholesterol, however, the effect of tegaserod may have 
been normalization of cholesterol exposure despite a continued high cholesterol diet. Note: Even though you found no statistical effect of tegaserod on gallbladder cholesterol in animals receiving high cholesterol diet, you have not tested whether or not gallbladder cholesterol remained elevated 
above baseline – i.e., above gallbladder cholesterol in non-treated animals on a low cholesterol diet. 
It appears unlikely that it remains significantly increased in tegaserod treated animals on high 
cholesterol diets. Therefore, instead of a reversal of the disinhibition, it seems as likely that tegaserod may have prevented the increased cholesterol necessary to produce the effect. Please 
consider these comments and reconsider tegaserod conclusions. 
6. The authors need to discuss the disparate effects of high cholesterol diet on gallbladder versus 
jejunal versus ileal muscarinic contraction. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
7. Methods; Experimental Animals: Considering these were wild animals used for experimentation 
within a month of capture, can you state anything about the uniform health of these animals, 
especially as relates to internal parasitism? Were animals given an anthelmintic and, if so, what? 
8. Methods; Experimental Animals, Page 6: “Animals randomly divided into four groups (19 in 
each)…”; so, this accounts for 76 of the 96 animals used. Please explain how the other 20 were 
used or make corrections as necessary. The very next paragraph does not explain which animals 
were used for the determination of hepatic and gallbladder bile cholesterol concentration? Please 
clarify. 
9. Cholesterol Concentrations in Hepatic and Gallbladder Bile, page 6: Please define “acute” in 
“acute terminal experiments in vivo…” Were these animals in addition to those used in the 
contractility experiments? Please clarify. 
10. First line of page 7: remove extraneous “(“. 
11. First sentence under Intestinal Contractility in vitro, page 7: (a) Please clarify. Were experiments 
started after 4 weeks on diet by testing 2 animals per diet group daily? (b) Please clarify how 
preparations from each animal were used. Was it one jejunum/one ileum for carbachol alone, one 
jejunum/one ileum for carbachol in the presence of TTX, one jejunum for carbachol in the presence 
of hexamethonium, leaving one jejunum for carbachol in the presence of scopolamine? In the 
results (page 10), however, it is stated that ileal contraction was not modified by hexamethonium. In 
which animals were the effects of hexamethonium studied on ileal segments? 
12. Intestinal contractility in vitro, page 7: (a) Please state temperature of tissue bath system. (b) 
Please clarify if bethanechol was added cumulatively or non-cumulatively? If the latter, in what order 
were concentrations added? (c) Please state how long antagonists were allowed to pre-incubate 
before constructing concentration-response curves? 
13. Intestinal contractility in vitro, page 7: 0.1 µM TTX is pretty minimal; 0.3 µM is generally needed 
in guinea pigs and at least 1 µM in mice and rats to reliably block nerve-mediated effects. Is there 
any reference that documents this concentration is sufficient to block nerve-mediated activity (MMC, 
peristalsis, or electric-field induced)? Nonetheless, you observed an effect that might best be 
qualified as highly sensitive to block by TTX. 
14. Reference [9] (top of page 8) is a study of the migrating myoelectric complex and does not 
involve stress measurements. Please provide appropriate reference. 
15. Page 8: Please fix subject verb agreement in second line: “…, the tissue preparation were…”. 
Please consider revising second sentence of Data Analysis. 
16. Data Analysis, page 8: Consider revising second sentence. Also, it should not be a mystery as to 
which statistical tests were used for each experiment. If not listed here, then it should be made clear 
in the Results or in the figure legends. [In my opinion, the cholesterol data should be tested with a 
two-way analysis of variance. The same could be done for predicted maximum effects for 
concentration-response curves. Addressing this additional comment is not essential as I don’t 
believe reanalysis would result in a change in the statistical differences found and not found. Power 
may have been a problem for the cholesterol measurements in the gallbladder.] 
17. Cholinergic Contractions of Jejunal Longitudinal Smooth Muscle, page 9: (a) Correct first 
sentence structure “…carbachol with increasing in tonic contractions…” and delete “optimal” as there is no indication as to why it is optimal; perhaps supramaximal. (b) Correct first sentence, second paragraph “…Newtons)/m2…” 
18. Effect of Action Potential Blockade on Carbachol-Evoked Contractions, page 9: (a) “Following 
TTX exposure …” is not correct. It was “In the presence of TTX …” (b) Second paragraph page 10: First sentence is awkward. Is the point that the TTX-induced enhancement of cholinergic jejunal 
contractions was not altered by pre-treatment with tegaserod in the low-cholesterol diet group? (c) 
Please make clear whether or not the effects of hexamethonium and scopolamine were tested on 
animals treated with tegaserod in the jejunum and ileum. 
19. Interactions…, page 11: Please correct text since you DO show the hexamethonium data in high 
cholesterol diet animals (Figure 3b). 
20. References are expected for line one of page 12: “…influences the affinity, binding capacity and 
signal transduction.” Especially references related to such effects on muscarinic receptors. 
21. Bottom of page 13: (a) Reference 41 only shows increased cholesterol content in gallbladder 
smooth muscle, which has reduced (not enhanced) contractions. It may be too much to assume that 
jejunal smooth muscle or jejunal myenteric neuron membranes also have increased cholesterol 
content. Direct exposure of myenteric motor neurons to lumen and absorbed substances does not 
seem likely. Note also that exposure to plasma cholesterol (previously shown to be elevated in these 
animals) would likely be similar for myenteric neurons of jejunum and ileum. (b) High cholesterol diet 
reduced an opposing inhibitory mechanism to the smooth muscle that is mediated by activation of a 
muscarinic receptor and involves opening of TTX-sensitive VGSCs. Might the effects of high cholesterol diet on this inhibitory mechanism be due to loss of receptors, loss of receptor transduction mechanism, and/or loss of inhibitory mechanism? 
22. Please clarify last sentence of page 13 that continues on to page 14. 
23. Figures (a) X-axis in all figures should be “log[Carbachol](M)” and not “[Carbachol].M”. (b) 
Correct supra-scripted end-bracket in Y-axis labels in all figures. (c) Figure 2: correct the spelling of 
tegaserod. (d) For future reference and potential comparison, you must present more detail of the data on ileal contractions. This might be easiest to achieve by inclusion of the concentration-response relationship figure. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
24. On page 2 within the Background of the Abstract: it is probably sufficient to use “5-HT4 receptor” 
instead of “5-HT4 serotonin receptor”. The latter is redundant and not used anywhere else in the 
manuscript. 
25. For clarity throughout the manuscript, consider consistent referral to the diets as low versus high 
cholesterol with definition given in the Methods. Within the abstract, for instance, the diets are 
referred to as low, 1% or enriched, and in the Results as “normal”. 
26. Eighth line page 5: It is not clear why “drugs used to treat this condition should have minimal 
impact on gallbladder contractility.” Would a drug that increased biliary emptying not be potentially 
helpful in decreasing the likelihood of stone formation? In either case, your data show a significant 
reduction in hepatic duct cholesterol with chronic tegaserod. Is that to be viewed as unfavorable? 
27. Regarding the normalization method: Although only a minor concern, are the authors certain that 
normalization using this formula remains appropriate after high cholesterol diet? In other words, is it 
known that the relationship of longitudinal smooth muscle area to whole preparation weight and length remains unchanged with diet? For instance, some diets reduce or increase intestinal length. While normalization of data is frequently helpful, do the basic relationships hold up if the data is not 
normalized in this way? 
28. In my opinion, there is no need to repeatedly state “significantly… (P<0.05)” throughout the 
manuscript when you’ve already defined significance as P<0.05 in the Methods. The exception 
would be for figure legends, which should be able to be read alone. 
29. It might be worthwhile noting in the discussion that these animals are expected to be 
hypercholesterolemic such that a difference between jejunum and ileum may have been luminal 
exposure to increased cholesterol or bile salts but probably not in exposure to plasma cholesterol. 
30. Figures 1 and 2 show the same data, but in a different combination. In my opinion, Figure 1 is not 
necessary. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Needs some language corrections before being published Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests. 
assistant