Dear Dr. ****:

Your manuscript: titled "Title' has been reviewed by a member of the Editorial Board and an expert external referee. I am sorry to inform you that both referees recommended that the manusctipt be declined. However, both referees found certain parts of the experirnental data to be interesting.

The most clear cut experiments in the manuscript are those concerned with the effect of A-kinase activation. Both referees noted the absence of any data on the effects of A-kinase activarion on the IP3 mediated oscillations . The referees also noted several negative comments (enclosed) . These included the inclusion of data that do not make substantial contributions to the manuscript (Figures I and 2) and several instances the description of the experiments and English usage could be substantially improved.

Despite these conditions, we would be happy to re-consider an extremely revised version containing additional data on A-kinase interactions.

Thank you for submitting this interesting work to the Journal of ****. We regret that it was not accepted in its present form and look forward to receipt of a revised version of the manuscript in the near future. If you do submit another version please return an original revised version along with two copies as well as a copy on computer disk.　This will facilitate the review process.

Yours sincerely,

Referee 1

The authors have made a study of the factors influencing ATP-mediated oscillations in K+ current using the nystatin-patch technique to introduce various molecules into the cells ( GTPgS , IP3 ) . The authors assume that the K+ current oscillations reflect Ca2+ changes and have attempted to discriminate between modelｓs of Ca2+ oscillations,

omments

1 , The authors have confirmed that the drugs do not directly modulate the K+- conductances. How was this done and where is the data reported ?

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

3. Figure 1 essentially shows that the oscillations in K+ current induced by ATP is not modified by ryanodine or procaine. Thie is negative data and can be included in the text.

4, Figure 2 This figure shows that thapsigargin and A23187 mobilizes Ca2+ and activates non-oscillatory currents . The novel point of these traces is not clear and this data could easily be omitted.

5 , p7 , second para, The three reasons given for the experimental finding in Figure 3 are phrased in poor english. Reasons 2 & 3 are simply not clear.

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

7 , The explanation behind the effect of the low concentration of thapsigargin ( Figure 3Bb ) cannot be as simple as stated by the authors because if the low concentration of thapsigargin was working by blocking the pump then it would have activated the K+ current .

8, p8. This is a rather one-sided view of the models proposed to account for Ca2+ oscillations, Wakui et.al., have shown that dialysis of cells with IP3S will promote oscillations ( Nature 339, 317, 1989 ) .

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

l0. Figure 5. If the PMA is washed out without applying the staurosporin does the system recover ? This control is not given in the figure.

11. Figure 7A. The current trace in Figure 7A is showing repetitive inward current spikes. Was the holding potential being varied in the experiment. These spikes are not visible in any of the other traces. The full experimental conditions should be documented in the Figure legends.

12. The authors should provide lieerature citations to show that protein kinase

c-activation stimulates Ca2+ accumulation by intracellular stores.

13. Figure 10, It would be helpful if the authors attached + and - to the arrows so to indicate whether the effector is acting in a positive or negative manner. The reader will then not have to refer to the text,

MS #****

Author: *****et al.

This study uses the nystatin pedorated patch clamp technique to study oscillations in a Ca2+ dependeni K+ cnannel In megakaryocytes. This assay is used to Investigate mechanisms and modulation of Ca2+ oscillatlons. This method has the advantage that it avoids the damping effects of the Ca2+ buffers normally used to measure Ca2+. albeit with the disadvantage that Ca2+ itself is not measured directly and that the changes might reflect Ca2+ changes below the cetl membrane that might not be applicable to the whole cell. Specific questlons addressed are: (i) Nature of intracellular Ca2+ pool; (ii) modulatlon by protein kinase C: and (iii) modulation by cAMP. The results Indicate that the oscillations involve an IP3 sensitive pool, not a classical Ca2+-induced Ca2+ release pool. Oscillations in IP3 concentration are not necessary, whereas Ca2+ reuptake into intracellular pools is necessary. Phorbol esters inhibit the oscillations. but the mechanism is unclear. ' The effect of PMA on responses to GTPgS and IP3 would be helpful to clarify the mechanism. Calmodulin antagonists interfere with the oscillations, apparently by altering reuptake, but it is difficuit to see the effects in the absence of expanded plots of individual Ca2+ spikes or time constants for reuptake. The ability of cAMP elevating agents to inhibit oscillations is clearly demonstrated using three different protocols (CTX, forskolin and IBMX). The effect appears to be on reuptake (time constants would be useful). The ability of these agents to affect the responces to GTPgS or IP3 would allow the site of action of cAMP to be assigned and avoid need for the question mark in the model (Fig. I O).

Specific Comments

1 , p. 5. Should state how it was confirmed that the drugs were not affecting the channel diretly. Was the Ca2+ sensitivity of the channel checked or just the current amplitude?

2 . In Flg. 2A, the response to ATP densensitize. How common ls this response?

3, The thapsigargin concentrations used in Figs. 2 and 3 are highly variable. It would be interesting to examine effects of submaximal thapsigargin on Ca2+ osciliations. Is the pattern similar to that for staurosporin or for W-7? A Iower thapsigargin is used in the low Ca2+ experiments (Fig. 3Bb) and also gives a sustained respense. Have the authors tried even lower concentrations?

4 , Fig. 1 . Explanation of the experiment not clear. ATP was applied transiently for 20-2S s. Was ryanodine present throughout? Are the 2 minute intervals between successive applications of ATP?

5. Fig. 4 is a nice demonstration that Ca2+ oscillations are not due to oscillations in IP3 concentration, since the much larger volume of the pipette compared to the cell should maintaln the IP3 concentration. Although IP3 appears to enter immediately (Fig. 4A), the series resistance of the pipette should be glven so that the diffusion time can be calculated by those interested. Better still, the authors can make the caiculation <see Pusch and Neher. 1988, PfluGers Arch. 411:204-21 1).

6. Staurosporin seems to enhance amplitude of IK,Ca (Fig, 5). Is this significant and reproducible?

7 , p. 8. Authors state that staurosporin inhibited 'the falling phase of IKca during ATP application in a concentration dependent manner, However, the time constant of the falling phase of each Ca2+ spike does not appear to be affected, This, though, is difficult to tell without expanded plots of single spikes. If there is a difference, some expanded plots shoutd be shown, or the numbers shown in the text.

8. Calmddulin antagonists also diminish the oscillations (Fig. 6)･ However, the effects ot W-7 and chlorpromazine are different. Chlorpromazine gives a plateau response, much like staurosporin. Howover, the control spikes seem to be becoming smaller, which is not the case in other control experiments. W-7 seems to have its largest effect immediatety, then the cell partially recovers its ability to pump Ca2+ out of the cytosol, Is this pattern also true of chlorpromazine? It should be if the mechanism of action (as calmodulin antagonists) is the same, This point should be addressed by showing more comparable ooncentrations of chlorpromazine (slightly lower) or dlscussing specificity of these agents. W･7 appears to increase the time constant for Ca2+ reuptake. This should be shown in expanded plots or the numbers given in the text.

9. GTPgS evokes oscillations, but unlike IP3, there is a delay (Fig. 7), Again, the series resistance should be given to allow comparison of diffusion times.

1 O , The effects of IBMX on frequency, Iatency and amplitude are shown. The reuptake time constant would be informative.

General

In general, this is an interesting study looking at Ca2+ oscillations. This experimental system seems an excellent system for investigating mechanisms and modulation of Ca2+ oscillations.The paper covers wide ground and provides some interesting data on modulation of Ca2+ oscillation, worthy of publication in J. ***. It was a little disappointing that a few loose ends were not tied up and that the discussion ended abruptly. Specifically, the effects of protein kinaso C and cAMP

dependent protein kinase modulation were only examined on the ATP-induced oscillations. The effeot of these agents on GTPgS and IP3 induced oscillations might allow a more exact positioning of the modulatory sites in the model. The paper would then represent a major contribution to our understandlng of the regulation of Ca2+ oscillations.