Dear Dr. ***,

Paper No ** 93/0357

The paper entitled "title" that you kindly submitted for publication in the ** Journal has now been considered by an external referee and a member of the Editorial Board. I regret to tell you that they were unable to recommend acceptance of the paper in its present form, for reasons given in the enclosed composite Editorial Report.

The reviewers found the paper of interest, but felt that it would be strengthened by additional data on several aspects (points 1-3), and that clarification of some other issues was required (points 4 and 5).

If you can deal satisfactorily with the points raised by the reviewers we would be happy to consider a revised version of your paper, although we cannot give a firm commitment to eventual publication at this stage. To facilitate further review, a revised manuscript should be accompanied by a letter detailing how you have responded to the individual points raised in the Editorial Report.

Yours sincerely,

Editorial Report

The paper describes the effect of imposed changes in cytoplasmic pH on free Ca-dependent oscillations induced by extracellular ATP in megakaryocyies. The data is in general clear and well presented.

Major points

1. Fig. I shows the time-course of BCECF-monitored pHi changes on a time-scale of tens of minutes. Yet in Figs. 2, 3 the effect on ATP-induced oscillations of NH4Cl (panel a) occurs within the first spike (1-3 seconds?), and NaOAc takes only 10 seconds to restore spiking. So we need a more detailed time-course of the BCECF signals than is provided by Fig. I . If, as seems likely, the oscillations are modified well before any BCECF-measurable change in pHi has occurred then interpretation of the papers' data become more tortuous.

2. Is the experiment on p 2-3 in which A23187 was used to raise Ca free to show that NH4Cl or NaOAc has no direct effect on the K channel valid? Perhaps A23187 could have effectively prevented any induced pHi changes, especially close to the plasma membrane. A BCECF measurement using non-fluorescent Bromo-A23 1 87 would help substantiate their claimed lack of direct effects of NH4Cl or NaOAc.

3. Of course, in view of the title which implies that pHi changes are changing cytoplasmic Ca (and not just the channel activity) some direct fura 2 measurements would be very persuasive.

4. While the pHi changes are measured in isolated cells, the effects of pHi changes are measured in cell-attached mode. To what extent is the cell-attachment to the patch pipette likely to modify the pHi responses?

5. It is quite difficult to compare the data of Fig. 2B, where there appears to be a rather atypical spike pattern with that of Fig. 3A. Was the concentration of IP3 maximal in 3A, because if so it may not have been possible for NaOAc to enhance the response?

Minor points

6. Summary: IP3 is inositol 1 4 5 trisphosphate, not phosphatidyl inositol 1 4 5 triphosphate - which would be a hypothetical lipid species.

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