Dear Dr ***
Ms ****: Title
Your paper has been seen by a reviewing Editor and an Expert Referee and I regret to inform you that it is not considered acceptable for publication in The Journal of *, at least not in its present form. The enclosed reports set out various criticisms of your work. The Reviewers feel that you may be able to revise your paper in away that deals satisfactorily with these criticisms, although at this stage we cannot qive you an undertaking that a revised paper would be ultimately acceptable for publication . If you feel that you can comply with these requests, then we would be pleased to give the revised version of your paper further editorial consideration. Please send two copies together with a covering letter briefly detailing the changes you have made in response to each of the points raised by the Revi ewers . I apologise for the length of time spent on reviewing your manuscript and hope you have not been inconvenienced.
Yours sincerely :

Referee 1
Ms ****
This Ms was submitted after Ms **** was rejected earlier this year. The new Ms represents a substantial improvement over the previous one, as several of the problems raised during the first round of refereeing have been adequately solved. Nevertheless two critical points still need to be clarified for the Ms to become acceptable for publication. The first point concerns the comparison between conventional whole cell recording (WCR) and perforated patch recording (PPR). On p. 11 it is claimed that "all the differences between WCR and PPR might be caused by the presence or absence of EGTA in the pipette solution". However the evidence backing this claim is weak. The O-EGTA experiments mentioned on p. 11 are not illustrated or quantified. This should be corrected. If the authors' hypothesis was correct then 0.3 mM EGTA would dampen the response to a considerable extent, suggesting that the intrinsic buffering power of megacaryocytes is remarkably low. Alternative possibilities would include an artefactual Ca rise linked to a higher leakage conductance in WCR. Finally, the sentence "But in this case, . . . solution" on p. 18 would suggest that in fact differences between PPR and WCR are not simply due to differential Ca buffering. The second point needing clarification concerns the pharmacology of the purinergic receptor. At present there is no way to exclude that the atypical pharmacological profile results of the superposition of two classical receptors, for example P2T and P2Y･ The authors should discuss this issue and at least attempt to resolve it. One important point to test, for example, is whether ******以下省略

Referee 2
This paper presents data of K.-current oscillations evoked by purinoreceptor stimulation in megakaryocytes. The current oscillations are abolished by pretreatment of the cell with BAPTA-AM, therefore the current oscillations reflect cytoplasrnic Ca2+ oscillation. The style of the paper has been changed and resubmitted. According to the authors' reply they have stressed the presence of novel type of purinoreceptor in megakaryocytes and the advantage of nystatin perforated patch technique in the resubmitted paper. I comment on these points. 1 ) The paper suffers from the point that the characterization of a type(s) of purinoreceptor is rather weak. Megakaryocytes has been reported to possess a type or types of purinoreceptor(s) stimulated with ADP (*****). So the point is whether ATP- and ADP-induced responses are really resulted from the stimulation of novel type of purinoreceptor. To conclude the receptor is novel, one has to have detailed characterization of it. First, do the authors think only one class of purinoreceptor present in megakaryocytes or more? In "Discussion" they mention that "Except the high affinity for ADP, . . . resembled P2Y" (P･20, Iine 4), indicating they think only a subclass of P2Y,'receptor exists. But in the other part, they mentioned that we mainly used ATP as an agonist ..., because ATP did not induce aggregatory changes of megakaryocyte" (in "Results", p.11, Iine 3 from the bottom). The sentence can be read as ADP induces aggregatdry changes but ATP does not. The paper by Levine & Nachimias in the reference reported that ADP induced shape changes in megakaryocytes. In view of this the resultant cellular effects are different between ADP- and ATP-stimulation though these agonists can induce K+_current oscillation. In addition, Ikeda et al. (J. physiol., 447:711-) have reported that ADP-induced rises in internal C ~2+are partly due to entry of external Ca, presumably through Ni -sensitive channels . In the present paper the authors conclude that "Ca,. channel blocker ***. These suggest that ATP action is different from that of ADP both from cell morphology and Ca-entry Overall, it raises a question whether ATP and ADP really stimulate the same one class of receptor. If not, the comparison of order of potency amongst the agonists is somewhat nonsense. The referee has an impression that two types of purinoceptor at least present in this cell type, and the confusion of the present paper is due to the lack of strict strategy to characterize types of purinoreceptors. , 2) The authors stress advantages of perforated patch method. Nobody can deny the usefulness of it if used properly. In the present paper, the authors describe the advantage of the method only from the aspect of Ca-chelating agents. It looks that blunted responses with the conventional whole-cell recording is due to types and doses of Ca-chelating agents, and/or balance between external chelating reagents and internal Ca-buffering molecules. ' To keep advantages of perforated patch method in this cell type, it is required to show advantages of it over the ***以下省略

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