Title: The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-
induced constipation
Version: 3 Date: 08 Nov 2023
Reviewer's report:
General comment: Improve English. Please proofread the whole manuscript and correct
grammatical and spelling errors.
Specific comments:
Title: Improve. The word "layer" in the title does not seem correct. I suggest changing it to
"plexus".
Abstract: well organized; some corrections are suggested below:
1. Define the abbreviations EM2 and #-FNA.
Keywords: Good
Abbreviations: the acronym OIC is repeated twice and corrects the full name of MRS2500,
which is [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate].
Introduction: Some observations and corrections are below:
1. Avoid using unnecessary abbreviations, such as AEs (Adverse events).
2. The authors mention that "ATP regulates the functional activity of the colonic P2Y1 receptors
by acting as a high-affinity ligand". However, ADP is 100 times more affinity to the P2Y1
receptor is 100 times than ATP; please update the bibliographic citation.
3. Please include the bibliographic citation: "Previously, we showed that the endogenous
receptor agonist of MOR, endomorphin-2 (EM2), could completely block fIJP without affecting
sIJP in colonic circular muscle cells, which was consistent with the effect of P2Y1 receptor
antagonist MRS2500 on colonic fragments".
Materials and methods: Some observations and corrections are below:
1. In the section Animal used, specify the total number of rats used in this study.
2. In the section Gastrointestinal motility, I suggest removing the numbers in parentheses at each
paragraph's beginning because it confuses the reader.
3. Please indicate the protein concentration loaded on the SDS-PAGE gel in the section Western
blotting. Also, indicate whether the transfer was done in humid, semi-humid, or dry conditions.
4. In the section Mechanical Test, Improve and detail the procedure description. For example,
specify the application times of the agonists and antagonists used, if washes were done and for
how long, if organ bath chambers were used, the brand of transducer used, and what recording
system they used.
Results: Regular, needs to be improved. Some observations and corrections are below:
1. Correct the legend of Figure 1; delete A-C after the first point and indicate what the white
arrowhead in the images means.
2. Why look for the colocalization of ATP with the MOR receptor? Please, could you explain?
3. The authors describe in the results that "The IF-based histochemical analyses revealed that
many P2Y1-positive nerve cells were aggregated in the colonic myenteric plexus to form ganglia
(Fig 1)". The term "many" is subjective;  I suggest quantitatively analyzing expressing P2Y1
cells by area.
4. Why was the expression of P2Y1 and MOR not analyzed in opioid-induced constipation mice?
5. The authors mention in the methods section that the traits of rat feces were scored according
to reference [16], but this quantitative analysis is not shown in the results. Please include a feces
score analysis graph in your study.
6. Figure 3B shows statistically significant differences in the protein expression of the P2Y1
receptor between the NCG and NSG groups. Please check the figure because there appear to
be no differences.
7. In the Figure 3 legend, the word "colonic" is repeated twice; please correct it.
8. Why was the myenteric plexus not used in Western blot experiments? Why the muscle layer
was used instead? Please, can you explain it to me?
9. You describe that the selective antagonist of MOR, #-FNA, significantly inhibited the effect
of EM2 by reducing the spontaneous contractive amplitude of distal colon segments. However,
in Figure 4A, it is seen that the amplitude of the contraction already decreased before applying
the #-FNA. To see whether the decrease in the amplitude of the contraction is due to MOR
response, the antagonist #-FNA alone should have been applied a few minutes before and then,
together with the agonist EM2, to ensure that the response of the active opioid receptor is being
blocked. That is, EM2--- #FNA --EM2+ #FNA.
10. In the methodology, you do not specify whether atropine, guanethidine, and substance P
were used to block cholinergic, adrenergic, and tachykininergic responses in order to ensure that
the inhibition of the amplitude of the contraction is given directly by the activation of the P2Y1
receptor with the ADP#S agonist and not in an indirect way. Could you please discuss why these
non-specific response blockers were not used?
11. Please describe in the results all the sections of Figure 4 because you only refer to Figure 4
in general but do not specify each section (A-D) in the text.
Discussion:
1. Please include the appropriate bibliographic citation in the following statement: "This was
consistent with the characteristics of the OIC fecals in previous reports."
2. The term "enteric muscle plexus" is incorrect; please correct it to the myenteric plexus. The
terms plexus and layer are managed as indistinguishable in the whole manuscript, which is
wrong. Please check the entire text and correct it.
3. Please include the appropriate bibliographic citation in the following statement: "Studies have
shown that MOR mainly regulates the movement of GI smooth muscle by modulating the release
of excitatory and inhibitory neurotransmitters, including ATP, CO, and NO."
4. On several occasions, you use the term "voluntary contractions of GI smooth muscle." What
are you referring to? Because voluntary contractions are of skeletal muscle.
Title: The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-
induced constipation
Version: 3 Date: 08 Nov 2023
Reviewer's report:
General comment: Improve English. Please proofread the whole manuscript and correct
grammatical and spelling errors.
Specific comments:
Title: Improve. The word "layer" in the title does not seem correct. I suggest changing it to
"plexus".
Abstract: well organized; some corrections are suggested below:
1. Define the abbreviations EM2 and #-FNA.
Keywords: Good
Abbreviations: the acronym OIC is repeated twice and corrects the full name of MRS2500,
which is [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate].
Introduction: Some observations and corrections are below:
1. Avoid using unnecessary abbreviations, such as AEs (Adverse events).
2. The authors mention that "ATP regulates the functional activity of the colonic P2Y1 receptors
by acting as a high-affinity ligand". However, ADP is 100 times more affinity to the P2Y1
receptor is 100 times than ATP; please update the bibliographic citation.
3. Please include the bibliographic citation: "Previously, we showed that the endogenous
receptor agonist of MOR, endomorphin-2 (EM2), could completely block fIJP without affecting
sIJP in colonic circular muscle cells, which was consistent with the effect of P2Y1 receptor
antagonist MRS2500 on colonic fragments".
Materials and methods: Some observations and corrections are below:
1. In the section Animal used, specify the total number of rats used in this study.
2. In the section Gastrointestinal motility, I suggest removing the numbers in parentheses at each
paragraph's beginning because it confuses the reader.
3. Please indicate the protein concentration loaded on the SDS-PAGE gel in the section Western
blotting. Also, indicate whether the transfer was done in humid, semi-humid, or dry conditions.
4. In the section Mechanical Test, Improve and detail the procedure description. For example,
specify the application times of the agonists and antagonists used, if washes were done and for
how long, if organ bath chambers were used, the brand of transducer used, and what recording
system they used.
Results: Regular, needs to be improved. Some observations and corrections are below:
1. Correct the legend of Figure 1; delete A-C after the first point and indicate what the white
arrowhead in the images means.
2. Why look for the colocalization of ATP with the MOR receptor? Please, could you explain?
3. The authors describe in the results that "The IF-based histochemical analyses revealed that
many P2Y1-positive nerve cells were aggregated in the colonic myenteric plexus to form ganglia
(Fig 1)". The term "many" is subjective;  I suggest quantitatively analyzing expressing P2Y1
cells by area.
4. Why was the expression of P2Y1 and MOR not analyzed in opioid-induced constipation mice?
5. The authors mention in the methods section that the traits of rat feces were scored according
to reference [16], but this quantitative analysis is not shown in the results. Please include a feces
score analysis graph in your study.
6. Figure 3B shows statistically significant differences in the protein expression of the P2Y1
receptor between the NCG and NSG groups. Please check the figure because there appear to
be no differences.
7. In the Figure 3 legend, the word "colonic" is repeated twice; please correct it.
8. Why was the myenteric plexus not used in Western blot experiments? Why the muscle layer
was used instead? Please, can you explain it to me?
9. You describe that the selective antagonist of MOR, #-FNA, significantly inhibited the effect
of EM2 by reducing the spontaneous contractive amplitude of distal colon segments. However,
in Figure 4A, it is seen that the amplitude of the contraction already decreased before applying
the #-FNA. To see whether the decrease in the amplitude of the contraction is due to MOR
response, the antagonist #-FNA alone should have been applied a few minutes before and then,
together with the agonist EM2, to ensure that the response of the active opioid receptor is being
blocked. That is, EM2--- #FNA --EM2+ #FNA.
10. In the methodology, you do not specify whether atropine, guanethidine, and substance P
were used to block cholinergic, adrenergic, and tachykininergic responses in order to ensure that
the inhibition of the amplitude of the contraction is given directly by the activation of the P2Y1
receptor with the ADP#S agonist and not in an indirect way. Could you please discuss why these
non-specific response blockers were not used?
11. Please describe in the results all the sections of Figure 4 because you only refer to Figure 4
in general but do not specify each section (A-D) in the text.
Discussion:
1. Please include the appropriate bibliographic citation in the following statement: "This was
consistent with the characteristics of the OIC fecals in previous reports."
2. The term "enteric muscle plexus" is incorrect; please correct it to the myenteric plexus. The
terms plexus and layer are managed as indistinguishable in the whole manuscript, which is
wrong. Please check the entire text and correct it.
3. Please include the appropriate bibliographic citation in the following statement: "Studies have
shown that MOR mainly regulates the movement of GI smooth muscle by modulating the release
of excitatory and inhibitory neurotransmitters, including ATP, CO, and NO."
4. On several occasions, you use the term "voluntary contractions of GI smooth muscle." What
are you referring to? Because voluntary contractions are of skeletal muscle.
Please review this paper.
user
Title: The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-
induced constipation
Version: 3 Date: 08 Nov 2023
Reviewer's report:
General comment: Improve English. Please proofread the whole manuscript and correct
grammatical and spelling errors.
Specific comments:
Title: Improve. The word "layer" in the title does not seem correct. I suggest changing it to
"plexus".
Abstract: well organized; some corrections are suggested below:
1. Define the abbreviations EM2 and #-FNA.
Keywords: Good
Abbreviations: the acronym OIC is repeated twice and corrects the full name of MRS2500,
which is [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate].
Introduction: Some observations and corrections are below:
1. Avoid using unnecessary abbreviations, such as AEs (Adverse events).
2. The authors mention that "ATP regulates the functional activity of the colonic P2Y1 receptors
by acting as a high-affinity ligand". However, ADP is 100 times more affinity to the P2Y1
receptor is 100 times than ATP; please update the bibliographic citation.
3. Please include the bibliographic citation: "Previously, we showed that the endogenous
receptor agonist of MOR, endomorphin-2 (EM2), could completely block fIJP without affecting
sIJP in colonic circular muscle cells, which was consistent with the effect of P2Y1 receptor
antagonist MRS2500 on colonic fragments".
Materials and methods: Some observations and corrections are below:
1. In the section Animal used, specify the total number of rats used in this study.
2. In the section Gastrointestinal motility, I suggest removing the numbers in parentheses at each
paragraph's beginning because it confuses the reader.
3. Please indicate the protein concentration loaded on the SDS-PAGE gel in the section Western
blotting. Also, indicate whether the transfer was done in humid, semi-humid, or dry conditions.
4. In the section Mechanical Test, Improve and detail the procedure description. For example,
specify the application times of the agonists and antagonists used, if washes were done and for
how long, if organ bath chambers were used, the brand of transducer used, and what recording
system they used.
Results: Regular, needs to be improved. Some observations and corrections are below:
1. Correct the legend of Figure 1; delete A-C after the first point and indicate what the white
arrowhead in the images means.
2. Why look for the colocalization of ATP with the MOR receptor? Please, could you explain?
3. The authors describe in the results that "The IF-based histochemical analyses revealed that
many P2Y1-positive nerve cells were aggregated in the colonic myenteric plexus to form ganglia
(Fig 1)". The term "many" is subjective;  I suggest quantitatively analyzing expressing P2Y1
cells by area.
4. Why was the expression of P2Y1 and MOR not analyzed in opioid-induced constipation mice?
5. The authors mention in the methods section that the traits of rat feces were scored according
to reference [16], but this quantitative analysis is not shown in the results. Please include a feces
score analysis graph in your study.
6. Figure 3B shows statistically significant differences in the protein expression of the P2Y1
receptor between the NCG and NSG groups. Please check the figure because there appear to
be no differences.
7. In the Figure 3 legend, the word "colonic" is repeated twice; please correct it.
8. Why was the myenteric plexus not used in Western blot experiments? Why the muscle layer
was used instead? Please, can you explain it to me?
9. You describe that the selective antagonist of MOR, #-FNA, significantly inhibited the effect
of EM2 by reducing the spontaneous contractive amplitude of distal colon segments. However,
in Figure 4A, it is seen that the amplitude of the contraction already decreased before applying
the #-FNA. To see whether the decrease in the amplitude of the contraction is due to MOR
response, the antagonist #-FNA alone should have been applied a few minutes before and then,
together with the agonist EM2, to ensure that the response of the active opioid receptor is being
blocked. That is, EM2--- #FNA --EM2+ #FNA.
10. In the methodology, you do not specify whether atropine, guanethidine, and substance P
were used to block cholinergic, adrenergic, and tachykininergic responses in order to ensure that
the inhibition of the amplitude of the contraction is given directly by the activation of the P2Y1
receptor with the ADP#S agonist and not in an indirect way. Could you please discuss why these
non-specific response blockers were not used?
11. Please describe in the results all the sections of Figure 4 because you only refer to Figure 4
in general but do not specify each section (A-D) in the text.
Discussion:
1. Please include the appropriate bibliographic citation in the following statement: "This was
consistent with the characteristics of the OIC fecals in previous reports."
2. The term "enteric muscle plexus" is incorrect; please correct it to the myenteric plexus. The
terms plexus and layer are managed as indistinguishable in the whole manuscript, which is
wrong. Please check the entire text and correct it.
3. Please include the appropriate bibliographic citation in the following statement: "Studies have
shown that MOR mainly regulates the movement of GI smooth muscle by modulating the release
of excitatory and inhibitory neurotransmitters, including ATP, CO, and NO."
4. On several occasions, you use the term "voluntary contractions of GI smooth muscle." What
are you referring to? Because voluntary contractions are of skeletal muscle.
assistant