Dear Dr. ***
Please find enclosed a revised manuscript entitled "Title" by Authors which we re-submit for publication in THE JOURNAL OF ****. Previously, the major objection to the manuscript related to the pH sensitivity of Fura-2. ******** this point, we have corrected our Ca2+ measurements for pH-induced changes of Kd in a representative experiment (Figure 9). However, the correction is, in general, unnecessary and does not influence the interpretation of experimental results.
On attached separate pages, we address item by item the specific criticisms of Review-ers I and 2. I believe that we have addressed all criticisms thoroughly and completely, and I hope you will now find the manuscript acceptable for publication. .
In closing, I regret not having returned the revised manuscript to you more quickly, but the return of Dr. *** to Japan after completing his postdoctoral fel]owship and the birth of a baby in my household delayed us somewhat.
I Iook forward to your favorable consideration.
Yours Sincerely,
RESPONSE TO REVIEWER 1
1. "the indicators used to measure intracellular Ca2+ are pH sensitive"
In the revised manuscript, we have careful]y eva]uated the effects of changes of pH･ on our calcium measurements. Figure 3 now presents actual measured changes of pH･ during our experiments. Figure 8 compiles existing data concerning the dependence of Kd on pH. Figure 9 shows cytosolic free Ca + after chemical hypoxia with and without a correction for the change of Kd caused by changes of pH･. This analysis demonstrates that errors in estimates of free Ca + are never more than 30 %. Thls is less than hormone-induced changes of Ca2+ and much less than the several fold increase expected by proponents of the Ca hypothesrs of cell injury. This interpretatlon is supported by our monensin experiments (Figure 12). With monensin PHI is clamped to pHo.' Nevetheless ATP depletion after metabolic inhibition caused little change of cytosolic free Ca2+ even though the effect of ***. we have chosen simply to present *** Because the effect of pH on estimates uncorrected for changes of pHi. This keeps the manuscnpt as short and concise as possible and avoids unnecessary confusion of the reader.
2. "Previous studies ... showed that HgCl2 had a profound effect [on pyridine nucleotide fluorescence], does 'chemical hypoxia' also have an effect .., if so this has to be shown"
The marked increases of Fura-2 fluorescence at Ca2+-dependent and independent wavelengths observed after HgCl2 and associated with pyridine nucleotide oxidation are not observed after chemical hypoxia. After chemical hypoxia, pyridine nucleotides become more reduced.**** but autofluorescence is so weak
3. "the figures are mislabeled"
Corrected.
4. 'The inositol phosphate measurements"
In some experiments, we measured inositol phosphate production by release of total "mono-, bis, tri- and tetra-phosphates (p. 7) from cells loaded with radiolabeled myo-inositol. Our technique, with minor modifications, is that described by lrvine et al. in 1985 in The Biochemical Journal and has been used frequently in studies published in The JOURNAL OF **** and elsewhere. Although techniques have developed to discriminate each polyinositol and its isomers, this original technique remains valid and is simpler and less expensive to app]y. The reviewer does not suggest that our measurements are incorrect or misleading in any way. We thank the reviewer for bringing the newer techniques to our attention. We will use them in the future.
5. "Does chemical hypoxia cause inhibition of hormone binding to heptitocytes and hence cause cessation of the oscillations?"
The effect of chemical hypoxia is the same, regardless of whether hormone agonists have already been bound to their receptor (e.g. compare Figure 6A with 6B-D). Like sub-strate binding to an enzyme, hormone binding to its receptor is believed to be an ATP-independent event. Many examples of hormone binding to receptors in isolated membranes show binding is not ATP-dependent.
RESPONSE TO REVIEWER 2
1. 'The intracellular concentration of fura2 is given as 190 ｵM and yet the author states that this would not dampen calcium oscillations. This is approximately a 10-20% increase in calcium binding components in the cell, so fura2 presence should not be taken so lightly"
The original manuscript was in error. 190 ｵM was the concentration of SBFI in the cells. Previously, we determlned that Fura-2 concentration in hepatocytes loaded under the conditions used was about 50 ｵM, corresponding to a 3-5% increase of intracellular calcium buffering capacity. We discuss this point because we do not take the issue lightly. The data indicate that Fura-2 causes almost no change in the calcium buffering capacity of the cells.
2. "The authors correct SBFI for [pH]i, yet do not do so for fura2"
In the revised manuscript, we have carefully evaluated the effects of changes of pHi On our calcium measurements. Figure 3 now presents actual measured changes of pHi durin our experiments**** Figure 9 shows ' ' , . . the change of caused by changes of pH･. This analysis demonstrates that errors in estimates of free Ca 2+ are never more than 30****
was to accelerate the onset of lethal cell injury. Because the effect of pHi On Ca + estimates was relatively small, in most experiments we have chosen simply to present estimates uncorrected for changes of pHi to avoid reader confusion and to keep the manuscript concise.
3. Typographical error on p. 11.
Corrected.
4. Mislabeled figures.
Corrected.