Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission“ by Khairullin et al. investigates purinergic modulation of ACh/ATP release from alpha-motoneuron endings resp. muscle contraction of the M. soleus or M. EDL using a myoelectrical approach. The nerve stump innervating the M. soleus/ M. EDL were stimulated by electrical field stimulation and force was recorded using an isometric mechanical activity sensor. To test a purinergic modulation of contraction force, the authors compared contraction force under control conditions with that of agonist/antagonist exposure. By using pharmacological blockers, the authors report that ATP or Adenosine, acting through P2 or A1 receptors, reduce contraction force.

The experimental approach of the manuscript seems overall good, however, some points need to be clarified:

 1) The authors state that their electrical stimulation paradigm specifically excited the nerve stump and not directly the muscle, hence any observation made in this manuscript is due to presynaptic ATP/Adenosine-induced modulation of ACh release (see Figure 5). However, the authors largely ignore the fact that cAMP signaling itself may also affect contractility (see PMID: 22354781). How can the authors rule out a postsynaptic mechanism of any of the substances applied? Have the authors tested whether the drugs used in their study potentially affect contraction force itself? In the discussion, the authors themselves state that “The mechanism of the inhibitory effect of ATP may be associated with a decrease in the release of Ca2+ from the sarcoplasmic reticulum, as was previously shown for ADP in the slow fibers of the rat soleus muscle [26]. Suramin, a non-selective P2 receptor blocker, abolished this action of ATP. Thus, it can be assumed that ATP exerts its inhibitory effect on rat phasic muscle contractions by influencing P2Y receptors.” (p.6, ll. 201-205)

2) The manuscript would greatly profit from example traces of contractions under the different conditions described.

3) How was contraction force assessed?

Editorial:

The paper is sometimes difficult to read and would at some instances profit from a more concise way of writing.

p.1, ll. 19-20: the word ‚‘is‘ is missing from the second main clause.

p. 1, ll. 21-23: please consider rephrasing this sentence.

p. 2 ll. 73-75: Did contractile reactions vary between control stimulation #1 and control stimulation #2? Were data points normalized to the mean contractile reaction of both control stimulations or to either control stimulation ( #1 or #2)?

 

Comments on the Quality of English Language

English grammar and sentence structure should be improved at some instances.

Author Response

Response to the Reviewer 1

 

Reviewer 1:

 

The manuscript entitled “Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission“ by Khairullin et al. investigates purinergic modulation of ACh/ATP release from alpha-motoneuron endings resp. muscle contraction of the M. soleus or M. EDL using a myoelectrical approach. The nerve stump innervating the M. soleus/ M. EDL were stimulated by electrical field stimulation and force was recorded using an isometric mechanical activity sensor. To test a purinergic modulation of contraction force, the authors compared contraction force under control conditions with that of agonist/antagonist exposure. By using pharmacological blockers, the authors report that ATP or Adenosine, acting through P2 or A1 receptors, reduce contraction force.

The experimental approach of the manuscript seems overall good, however, some points need to be clarified:

 

Our Response:

Thank you for your positive consideration of our paper. We have revised it according to the Reviewers’ comments and suggestions and hope that in a revised form the manuscript will be accepted for the publication in the CIMB.

Reviewer 1:

 

• The authors state that their electrical stimulation paradigm specifically excited the nerve stump and not directly the muscle, hence any observation made in this manuscript is due to presynaptic ATP/Adenosine-induced modulation of ACh release (see Figure 5). However, the authors largely ignore the fact that cAMP signaling itself may also affect contractility (see PMID: 22354781). How can the authors rule out a postsynaptic mechanism of any of the substances applied?

Our Response:

The cAMP signaling pathway that the reviewer mentions is intracellular, but in our experiments we are talking about the effects of extracellular ATP and adenosine. Of course, metabotropic postsynaptic P2Y receptors may be coupled to this signaling pathway. But in our works PMID: 27448234 and PMID: 30677146, we showed the absence of postsynaptic effects of ATP (at a temperature of 37^o) in the model of carbachol-induced contractions.

 

Reviewer 1:

Have the authors tested whether the drugs used in their study potentially affect contraction force itself? In the discussion, the authors themselves state that “The mechanism of the inhibitory effect of ATP may be associated with a decrease in the release of Ca2+ from the sarcoplasmic reticulum, as was previously shown for ADP in the slow fibers of the rat soleus muscle [26]. Suramin, a non-selective P2 receptor blocker, abolished this action of ATP. Thus, it can be assumed that ATP exerts its inhibitory effect on rat phasic muscle contractions by influencing P2Y receptors.” (p.6, ll. 201-205)

 

Our Response:

Yes, we checked whether the drugs used in our study affected the force of contraction itself. In each series of experiments, we assessed the effect of P1 and P2 receptor antagonists (Figures 3 and 5) as well as presynaptic inhibitors (Figure 6-7).

 

Reviewer 1:

 

• The manuscript would greatly profit from example traces of contractions under the different conditions described.

Our Response:

Thank you for this suggestion. We have prepared 2 new Figures to the MS with original traces for m.soleus and inserted them to the body of the paper. Similar traces can be included for m.EDL if the Reviewer 1 or the Editor will say they are needed.

 

Figure 1. Original traces of contractile responses of the isolated rat m. soleus evoked by electrical field stimulation (0.1 Hz, 0.5 ms, 10 V) in control (a) and in the presence of ATP (100 μM, b), suramin (100 μM, c), a combination of ATP and suramin (both at 100 μM, d).

Figure 3. Original traces of contractile responses of the isolated rat m. soleus evoked by electrical field stimulation (0.1 Hz, 0.5 ms, 10 V) in the control (a) and in the presence of adenosine (100 μM, b), 8-sulfophenyltheophylline (100 μM, c), a combination of adenosine and 8-sulfophenyltheophylline (both at 100 μM, d).

 

Reviewer 1:

• How was contraction force assessed?

 

Our Response:

Contraction forces were recorded using a Linton FSG-01 isometric mechanical activity sensor (Great Britain), and the analog signal was digitized and processed using a Biopack MP100WSW data acquisition system (USA).

Reviewer 1:

Editorial:

The paper is sometimes difficult to read and would at some instances profit from a more concise way of writing.

Our Response:

Thank you for your comments. We gone through the text of the manuscript several times ourselves and asked one of our colleague with good English to help us. We found that we used too many commas in the text (which is a standard for Russian texts), we found some misprints and wrong style sentences. We hope that we have corrected the text to the needed standard, including the lines you have mentioned below.

Reviewer 1:

p.1, ll. 19-20: the word ‚‘is‘ is missing from the second main clause.

Our Response:

            Thank you, corrected.

Reviewer 1:

1. 1, ll. 21-23: please consider rephrasing this sentence.

Our Response:

            The sentence is rephrased to:

We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A1 receptors while the positive action is associated with the stimulation of adenosine A2A receptors, activation of protein kinase A and opening of L-type calcium channels.

Reviewer 1:

1. 2 ll. 73-75: Did contractile reactions vary between control stimulation #1 and control stimulation #2? Were data points normalized to the mean contractile reaction of both control stimulations or to either control stimulation (#1 or #2)?

Our Response:

In most of the experiments the control contractile responses 1 and 2 were not different, in rare cases – the second response was a bit more or a bit less than the first one. However, in all experiments we used an average of 1 and 2 as an initial control contraction.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, the authors using a mechanomyographic method analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat soleus and EDL muscles.

The idea of this study - is interesting; but his manuscript needs some improvements and corrections before publishing may be possible.

 

General points:

Please add a list of abbreviations before References section to your manuscript.

 

Special points:

Abstract

Line 14: please write out: EDL

 

Keywords

Please add also to keywords: rats, P1-receptors.

 

Introduction

Lines 30-42: please add multiple references at the end of each these sentences.

Lines 43-48: please describe all these studies very exactly

 

Materials and Methods

First of all, please add the organization number, date and the protocol number of the permission for all your experiments.

Please add as a Figure 1 the time line for all your experiments used for this study.

Line 54: please add the old of rats used in your study.

Please add the number of rats and sex used for each method in your study.

 

What about this separate experiment? Please add the number of rats and sex used for each method in your study.

Lines 77-81: please write out all abbreviations in the text of your manuscript. 

 

Please add to each method the appropriate references according to which group or publication you did this method.

 

Results

Figures 1-4: please describe all groups of the animals very exactly (right side of all your Figures 1-4) in the Materials and Methods section of your manuscript.

 

Conclusion

Please add more Conclusion and Future Perspectives section to your manuscript.

 

Author Response

Response to the Reviewer 2

 

Reviewer 2:

In this work, the authors using a mechanomyographic method analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat soleus and EDL muscles.

The idea of this study - is interesting; but his manuscript needs some improvements and corrections before publishing may be possible.

 Our Response:

Thank you for your positive consideration of our paper. We have revised it according to the Reviewers’ comments and suggestions and hope that in a revised form the manuscript will be accepted for the publication in the CIMB.

Reviewer 2:

            General points:

Please add a list of abbreviations before References section to your manuscript.

Our Response:

Thank you for this suggestion. We have prepared the list of abbreviations and inserted it to the MS before the reference list.

Reviewer 2:

Special points:

Abstract

Line 14: please write out: EDL

Our Response:

            Thank you, corrected.

Reviewer 2:

Keywords

Please add also to keywords: rats, P1-receptors.

 Our Response:

            Thank you, included.

 

Reviewer 2:

Introduction

Lines 30-42: please add multiple references at the end of each these sentences.

 Our Response:

Thank you, the text is modified accordingly

Reviewer 2:

Lines 43-48: please describe all these studies very exactly

Our Response:

Here we did not understand the Reviewer. Should we give more references for these works or we need to give more details of the studies mentioned?

Reviewer 2:

Materials and Methods

First of all, please add the organization number, date and the protocol number of the permission for all your experiments.

Our Response:

            Thank you for this suggestion. The following sentence is added to this section:

The animal study protocol was approved by the Ethics Committee of Kazan State Medical University (protocol code 106/4, from 14 December 2021).

Reviewer 2:

Please add as a Figure 1 the time line for all your experiments used for this study.

Our Response:

            Thank you for this suggestion. The Figure is prepared and inserted to the paper.

 

Reviewer 2:

Line 54: please add the old of rats used in your study.

Our Response:

            We used 4-5 months old rats. Correction is made accordingly.

Reviewer 2:

 

Please add the number of rats and sex used for each method in your study.

Our Response:

            We used only male rats.

The number of animals used (N) were the following: suramin – 6, 8-spt – 5, DPCPX – 5, N-ethylmaleimide – 5, Rp-cAMP – 5, Nitrendipine – 5, Chelerythrin – 4, d-tubocurarine – 3.

But these are general number of the animals, from each animals we took both m.soleus and both m. EDL. In all experiments we have done controls, as such all results are the average of certain number of muscle sample used (n).

We think that it is not practical to include these numbers (N) in the text, since after the result figures we always shown the number of muscle samples used (n),

Reviewer 2:

What about this separate experiment? Please add the number of rats and sex used for each method in your study.

Our Response:

In the experiments with d-tubocurarine we used 3 male rats. We added the number of rats to this section.

Reviewer 2:

Lines 77-81: please write out all abbreviations in the text of your manuscript. 

Our Response:

            Corrected accordingly.

Reviewer 2:

Please add to each method the appropriate references according to which group or publication you did this method.

Our Response:

            We added the following sentence and reference to the Materials and Methods section:

The experimental procedures were made as described earlier in [12]. Briefly the isolated muscles were fixed vertically…

Reviewer 2:

Results

Figures 1-4: please describe all groups of the animals very exactly (right side of all your Figures 1-4) in the Materials and Methods section of your manuscript.

 Our Response:

            The following sentence was added to the Materials and Methods section:

The animals were divided into 8 groups: “Suramin” - 6 rats, “8-spt” - 5 rats, “DPCPX” - 5 rats, “N-ethylmaleimide” - 5 rats, “Rp-cAMP” - 5 rats, “Nitrendipine” - 5 rats, “Chelerythrin” - 4 rats and “d-tubocurarine” - 3 rats.

Reviewer 2:

Conclusion

Please add more Conclusion and Future Perspectives section to your manuscript.

Our Response:

The following sentence was added to the Conclusion section:

The findings reveal potential targets for pharmacological intervention. However, the situation is not yet completely clear, and the purine signaling involved in the above-mentioned effects requires further comprehensive studies.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all concerns raised during the revision process. I have no further concerns and agree to a publication.

Author Response

We thank the Reviewer for the positive decision on our paper. 

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for ll your corrections.

You said: The presynaptic inhibitory effect of ATP and adenosine in the neuromuscular synapse has been described mainly in preparations of cold-blooded animals, while studies have been carried out only in fast-phase skeletal muscles [8–10]. In those few works [11], where experiments were performed on warm-blooded animals, the experiments were carried out on the synapses of the diaphragm, which cannot be considered as a typical effect of purines on all phasic skeletal muscles of warm-blooded animals.

Once again, please give more details of all these mentioned studies. 

Author Response

Response to the Reviewer 2 report (round 2)

 

Reviewer 2:

 

 

Comments and Suggestions for Authors:

Thank you for ll your corrections.

You said: The presynaptic inhibitory effect of ATP and adenosine in the neuromuscular synapse has been described mainly in preparations of cold-blooded animals, while studies have been carried out only in fast-phase skeletal muscles [8–10]. In those few works [11], where experiments were performed on warm-blooded animals, the experiments were carried out on the synapses of the diaphragm, which cannot be considered as a typical effect of purines on all phasic skeletal muscles of warm-blooded animals.

Once again, please give more details of all these mentioned studies. 

 

Our response:

Sorry for not clarifying this point in the first round. We rewrote this paragraph and inserted it to the text. We also added additional reference [12] to the list.

            Revised paragraph:

            The presynaptic inhibitory effect of adenosine on the amplitude of evoked phasic endplate currents was established in lake frog muscles [8]. Under similar conditions the inhibitory presynaptic effect of ATP was demonstrated [9]. At the same time, both pre- and postsynaptic potentiating effects of ATP were established on 2-week-old Xenopus tadpoles and in cell cultures of myocytes [10]. Much less know about action of purines on muscles of warm-blooded animals. Experiments on synapses in the rat diaphragm showed a presynaptic potentiating effect of activation of P2 receptors leading to facilitation of the release of acetylcholine [11] and later the inhibitory effect of adenosine and ATP on end plate potentials was demonstrated at the same object [12]. It is clear that further evaluation of the effects of these purines on other skeletal muscles of warm-blooded animals is needed.