Dear Dr. ***

Your manuscript no. M1716, entitied: Title

has been evaluated by the referee(s), who raised some points that are detailed on the enclosed evaluation sheet(s).

We are of the opinion that this manuscript can be accepted for publication in the Journal of *** MOLECULAR PHARMACOLOGY Section after minor revision.

We would appreciate it if you would modify the manuscript according to the suggestions made by the referee(s). Also, we inform you that, if a checklist is included, the points herein should be taken care of.

Please reply to the comments of the referee(s) and indicate which of the required changes have been made and where these changes are to be found in the revised manuscript (page and line). We also ask you to give the reasons that led you to reject some of the suggested changes.

Would you please send us the manuscript in duplicate, i.e. the entire revised manuscript and one copy thereof, including the complete set of illustrations and list of references for the manuscript and for the copy. Your reply to the comments made by the referee(s), also in duplicate, should accompany the manuscri pt.

Yours sincerely,

Referee #1

The paper by .***et al describes the effects of the proton ionophore FCCP on megakaryocytes isolated from rat bone. The major aim was to investigate the modulation of calcium oscillations in these cells by FCCP. As an indication of calcium concentration they use the evoked calcium dependent potassium current, measured under volrtage damp conditions. This is a valid and commonly used technique. though not necessariiy without interpretation problems as the authors indeed encounter in this study. The main reason may be that as long as the oscillations in calciurrL are slow and returrLing to tyhe same basehrLe level. the potassium current is a good interpretable indicator. When baseline calcium shifts or the osallations are very fast. the potassium signal could be more difficult to relate to the underlying changes in calcium. The authors might want to address these problems in their discussion. The papers contains a nice series of experirrtents and valuable data.

Minor points:

The A23187 experiments should not be mentioned in the abstract. since the results were not part of this study

There are several referencing problems (Berridge 1988; 'LJneyairL:a 1993a. Sc tz. 1989).

It must be possible to essess the modulation of FCCP in a more quantitative way (either frequency of oscillation or amplitudes) and give the readers a better statiscal indication of the significance of the observations.

This referee would suggest to change the tittle of the paper into one more closely related to the actual findings. FCCP modulation of calcium oscinations in.....?

The authors should adapt the last line of the discussion into a better evaluation of ･their results.

Referee #2

This manuscript describes a series of observations regardlng thc effeets of a protonophore on a Ca2+_activated K+ currrentt which was used as an index of fluctuations in free intracellular Ca2+. The issue is a timely one since Ca2+ oscillations are not wcll understood and the role of the ER H+ gradient in regulating the activity of the ER Ca2+_ ATPase is not yet fully characterized. The studies appear to be carefuny done and the effects of the protonophore FCCP are quite clear. The interpretation of the resutts is very complicated, but the authors have discussed most of the possibilities thoroughly. Their final conclusion regarding the use of protonophores to probe Ca:2 + regulafon is probably correct, at least given our current level of understanding . The major revision that is needsi is to correct several grammatical problems . These have been indicated directly in the manuscript so that they can be easily corrected, but there is a substantial number ef these that need attention '

,