June 1992
Distributing Editor
Dear Dr. ****
Thank you for your Fax with regard to our manuscript. And I am sorry to have troubled you. Enclosed our address to the specific criticisms of a Reviewing Editor and an Expert Referee to MS ****, and a copy of comments we got previously. The same report was sent to you by Fax already. I am looking forward to your favorable consideration.
Yours sincerely,
RESPONSE TO EDITOR
1. "These results are not really original since *** (1990) demonstrated almost identical results on response to ADP."
The results are not identical to ***'s because our currents are oscillatory ones, though quite similar. We think it is important to demonstrate the property of K+ channel is the same regardless of the recording method. Especially, we attempted to compare the nystatin perforated whole-cell patch-clamp method to conventional whole-cell method in our new manuscript. Thus, our manuscript includes these results again.
2. "BAPTA-AM was just added to the external solution. This is not a good procedure ...BAPTA-AM and some of the diffusible molecules ... produced unwanted side effects."
The method to load BAPTA-AM was described more clearly. It was not fully enough described in previous manuscript. In addition, we confirmed that another acetoxymethylester compound which did not alter intracellular Ca2+ concentration (SBFI-AM) had no effect.
3. "p.10 and Fig. 5: ..."
Fig. 5 was deleted as we consider these results were not necessary.
4. "it should have seen whether monophasic responses could be obtained in your hands ..."
5. "Ca is known to bind ATP tightly. It appears possible that results such as shown could simply be the consequence of a selectivity of the agonist binding site of this response for ATP4-."
These are major points of our new manuscript. We performed some experiments as you recommended and get interesting results. The answer to these comments is our new manuscript. We are grateful to you for these criticisms.
6. "Table 1 is unnecessary..."
Deleted and corrected as suggested.
7. "quinine and quinidine are notoriously NOT specific for K channels."
We have changed the explanation.
8. "Quotation of Wakamori et al. is not relevant to the present work."
Exchanged with relevant one.
9. "Quote Kawa on presence of Ca oscillations."
Quoted as suggested.
10. "In the figure legend ... I am not sure of what is meant by this sentense,..."
Our description was poor, thus we added the explanation in both figure legend and text.
11. "I assume that "10 mM ATP" should read "10 mM ATP". It is unclear to me what the numbers in the "pD2" column mean."
We are sorry for such an easy mistake. It was corrected. And the definition of pD2 was given in methods.
12. "There is no reason to attack Rooney et al. ...
Differential binding of Ca to ADP and ATP may account for part or all of the difference in potency between these two agonists."Changed as suggested. ADP had stronger effect than ATP even in the absence of Ca2+ and/or Mg2+, thus, the above mentioned possibility (especially the latter) was denied.
RESPONSE TO EXTERNAL REFEREE
Thank you for your comments.
Major points:
Our previous manuscript was not enough to clearly demonstrate important points of our findings. You were quite right to feel dissatisfied. Then we have changed the construction of our manuscript and added new findings. Our new manuscript contains demonstration of advantage of nystatin perforated whole-cell patch-clamp method in addition to novel type of purinoceptor on megakaryocyte. This manuscript will be worth to be read for many physiologist utilizing conventional whole-cell patch-clamp technique. We think, therefore, new manuscript will be more satisfactory than previous one.
Minor comments:
1. "In Methods, it is preferable to describe details of perforated patch technique ..."
Corrected as suggested.
2. "In Results, ... the reviewer really does not understand why the authors need to eliminate external Cl and/or NaCl ..."
Deleted these results as we consider them unnecessary.
3. "the figure (Fig. 6) is better to contain data recorded in the absence of external Ca2+ and ..."
The results appeared in Fig. 6 in previous manuscript were re-considered after comments by Editor, and presented again to suggest that the true agonist might be ATP4-.
4. "what is pD2?"
We described the definition in Methods.
5. "The authors used 10 mM-ATP for the normalization"
It was mistyped. We used 10 µM-ATP.
6. "the sentence, "Thus, it seems ... function of Ca2+ sensor", is not quite certain."
The paragraph including this sentence was deleted.
7. "the sentence starting with "As megakaryocyte ...", is again not certain."
The sentence was deleted. And we reconstruct the discussion as suggested.