Dear Dr. ***:

We submit a manuscript entitled "Title" by Authors, which is the revised version of our previous manuscript with MS #M2-1759. Enclosed an original manuscript along with two copies and a copy on computer disk. The 3.5 inch floppy disk was formatted by Apple Macintosh computer, and the application used was Microsoft Word. In addition, the answers to the comments raised to M2-1759 were on attached separate pages. I Iook forward to your favorable consideration.

Sincerely yours ,

Answers to the comments by Referee 1

Thank you for your kind comments. We deleted Fig. I and 2 of previous paper and added new figure as Fig.8. Thus, the Fig. No. changed. Fig. 1-7 in revised version correspond to Fig. 3-9 in previous manuscript. Fig. 9 in revised version corresponds to Fig. 10 in previous one.

1 . The authors . . . drugs do not directly modulate the K+ conductances . . .

We added the explanation in the text (p6, first para.). And the current traces to show the results were attached to this letter.

2. Ref.4 is . . . A copy of this manuscript (or the summary page) must be provided to the reviewers.

We changed the ref. to already published one (pl5).

3. Figure 1 . . . This is negative data and can be included in the text.

We deleted the figure and corrected the text as you suggested (p6-7).

4. Figure 2 . . . this data could easily be omitted.

Deleted as you suggested.

5. p7, second para . . . are phrased in poor English . . . .

We improved the discussion (p8, first para).

6. The final sentence on p7 is also very poor English.

We deleted the sentence as it is not necessary.

7. The explanation behind the effect of the low concentration of thapsigargin

Explanation was added (p8, second para).

8. p8. This is a rather one-sided view of the models . . .

Discussion was improved as suggested (p9, Iine 7-16).

9. Figure 5. Data showing that staurosporin effects are irreversible are not presented and should be indicated as such in the text.

This was indicated in the text (p9, second para).

10. Figure 5. If the PMA is washed out without applying the staurosporin does the system recover? . . .

We added the control current trace in new Fig.3 as suggested.

11. Figure 7A. The current trace in Figure 7A is showing repetitive inward current spikes.

This was our mistake. The current trace mentioned was the one with step-pulse. In this manuscript, such experiment is not adequate. Thus, the trace was exchanged to the proper one.

12. The authors should provide literature citations to show that protein kinase C-activation stimulates Ca2+ accumulation by intracellular stores.

We don't know any report to show PKC-activation results in Ca2+ accumulation by intracellular sores. Please tell us if you know. Instead, as we think platelet plasma membrane is equivalent to intracellular membrane of megakaryocyte (demarcation channel), we cited a literature to show PKC-activation stimulated Ca2+ efflux from platelet (plO, 127).

13. Figure 10. It would be helpful if the authors attached + and - to the arrows . . . .

The figure was changed as suggested.

Answers to the comments by Referee 2

We deleted Fig. I and 2 of previous paper and added new figure as Fig.8. Thus, the Fig. No. changed. Fig. 1-7 in revised version correspond to Fig. 3-9 in previous manuscript. Fig. 9 in revised version corresponds to Fig. 10 in previous one.

Specific Comments

1 . p.5 . Should state how it was confirmed that the drugs were not affecting the chaunel directly. . . .

We added the explanation in the text (p6, first para). And the current traces to show the results were attached to this letter.

2. In Fig. 2A, the response to ATP desensitizes. . . .

Fig.2 was deleted. And the phenomena was not ATP desensitization. This was because K+ current was maximally activated by A23187, thus the successive application of ATP could not cause any response.

3 . The thapsigargin concentrations . . .

Our previous explanations were not enough. We corrected them (p8, second para). You might misunderstand the effect of 100 nM thapsigargin, because the drug itself does not induce any response at the concentration range between 10 and 300 nM. Sustained response was obtained when thapsigargin was applied in combination with 10 ｵM of ATP.

4. Fig.1 . Explanation of the experiment not clear . . . .

Fig. I was deleted. We improved the explanations for other experiments (See legend for new Fig. 1).

5. Fig.4 is a nice demonstration . . . series resistance of the pipette should be given so that the diffusion time can be calculated . . .

The diffusion time constant was calculated according to your suggestion and added discussion about the result in other part of the text (p 1 1 , second para).

6. Staurosporin seems to enhance amplitude of IKCa (Fig.5). . . .

This is neither significant nor reproducible (New Fig. 3).

7. p.8. Authors state that staurosporin inhibited "the falling phase of " However, the time constant of the falling phase of each Ca2+ spike does not appear to be affected. . . .

We used the word "falling phase" to mean recovery of the current to the basal level. This word was not appropriate and lead to misunderstanding. Thus the discussion was changed by using other expression (p9, second para) .

8. Calmodulin antagonists also . . . the effects of W-7 and chlorpromazine are different. ... W-7 seems to have its largest effect immediately, then the cell partially recovers its ability to pump Ca2+ out . . . W-7 appears to increase the time constant for Ca2+ reuptake . . . .

You are right. Chlorpromazine inhibits both CaM and K+ conductance. Thus we changed the figure to that of trifluoperazine. Trifluoperazine did not inhibit the K+ conductance. See Fig. 4B in revised text. The first spike of oscillatory IKCa is the largest one, and we think the amount of Ca2+ released should also be the largest. Thus, the inhibitory effect on Ca2+ pump looks largest at first. We don't think, therefore, the cell recover its ability to pump Ca2+ out of the cytosol. The time constant for each current varies from cell to cell. It may indicate some properties about Ca2+ movement, but in this report we payed attention only to whether the current recovered to the basal level or not to simplify the problem. The increase or decrease in time constant would be considered in other work.

9. GTP-Y-S evokes oscillation, but unlike IP3, there is a delay (Fig. 7). Again, the time constant would be informative.

Corrected as suggested (p11 , second para).

10. The effects of IBMX on . . . . The reuptake time constant would be inf ormative.

We don't think the reuptake time constant is necessary because it is not altered by IBMX. And we did not add it because of the same reason explained in comment 8.

General

Thank you for your kind comments that improve our manuscript. As you mentioned the major point is the lack of the data on the effects of PKA and PKC on IP3-induced oscillation, we added the data showing that PKA activation inhibited IP3-induced oscillation (Fig.8). But we could not at present show the effect of PKC on IP3-induced oscillation. As PKC is activated by agonist, the same concentration of PMA or staurosporin did not exert the same effects on IP3-induced and agonist-induced oscillation. This theme needs further investigation. It requires more long time.

Another point is about our poor English. Sorry for our lack of ability to write accurate English. We corrected the sentence pointed out by you.