Dear Dr. ***

Re: paper No ** 93/0357

Thank you for your letter dated 19 APR 1993, and please find enclosed a manuscript entitled "Title" by Authors, which is the revised version of our previously submitted manuscript (** 93/0357). The answers to the comments raised in the Editorial Report are as follows.

l. ... we need a more detailed time-course of the BCECF signals than is provided by Fig. I .

We added new data showing detailed time-course of pHi Change as Fig. I B. As the minimal recording interval is 2 s in our experimental system (mentioned in the text p5, first para), this is the best we can do. But 2 s interval prLust be sufficient to show that the oscillations are not modified well before any BCECF-measurable change in pHi has occurred.

2. ... A BCECF measurement using non-fluorescent bromo-A23 187 would help substantiate their claimed lack of direct effect of NH4Cl or NaOAc.

We performed experiments using ionomycin instead of A23187 and confirmed that the results were the same. The word 'A23187' in the text, therefore, was changed to ionomycin (p6,frrst para). In addition, we confirmed that the pHi change could be induced in the ionomycin-treated cells as control cells. The result was not seen in the manuscript but please refer Ref. I attached to this letter.

3. ... some direct fura 2 measurements would be very persuasive.

The megakaryocyte is very sensitive to Ca2+_chelating agents and after fura 2 Ioading, the oscillatory response disappear. Please see Ref. 2. In fura 2 Ioaded cells, the ATP-induced K+ current became small and transient (A), and fluorescent signal was also transient (B). The oscillatory K+_current can be obtained by reducing the amount of fura-2, but in such condition, the fluorescent signal cannot be detected. Thus, at least at present, we cannot perform direct fura 2 measurements in megakaryocyte. To answer the comment, therefore, we changed our title "cytoplasmic Ca" to "Ca-dependent K+-current" and described the feature of megakaryocyte in the text (p8). We do not think this change means the change in the conclusion of the manuscript. The Ca2+-activated K+-current reflect the cytoplasmic Ca2+ with high sensitivity.

4. ... To what extent is the cell-attachment to the patch pipette likely to modify the pHi responses?

The attachment of the glass electrode itself had no detectable effect on pHi change. It is not surprising, bccause intracellular circumstances in nystatin-perforated whole-cell recording mode are mostly intact. The effect on the cell may be as small as the effect given by glass coverslips under the cell. The data are not shown, but a comment was added in the text (p.5, frrst para).

5. ...Was the concentration of IP3 maximal in 3A, because if so it may not have been possible for NaOAc to enhance the response?

We exchanged the figure to the data obtained by lower concentrations of IP3. The new Fig. 3A may be better than the previous one.

6. Summary: IP3 is inositol 1,4,5-trisphosphate, not phosphatidyl inositol 1,4,5-triphosphate- ...

It was our mistake and corrected as suggested.

7. Fig.5 is superfluous and can be stated briefly in the text.

Fig. 5 was deleted as suggested.

We hope our responses are satisfactory ones for you. We are looking forward to your favourable consideration.

Yours sincerely,