Title: Echinacea-induced Cytosolic Ca2+ Elevation in Hek293

Background -The first sentence is too generic and not strickly linked to the aim of the paper. We have removed the first sentence in the abstract as suggested. -In the last sentence the term “hypothesize” is not correct, but it must be changed with “show” or another similar. We have changed the term “hypothesize” to “show”. -Line 11: change “specialized” with a more appropriate adjective (here and in other parts of the paper) We have changed the term “specialized”. Conclusion -The last sentence must be erased, since too generic Background -Line 16 page 5: this sentence create confusion in this point. The rationale in the background is better comprehended by-passing this sentence. -Idem as above for the two sentences page 6 lines 6-12. -Last sentence: The “wide use as a transfection model for receptors” do not justify the choice of this cell line for the aim of the paper We have removed all the above mentioned sentences. Methods -In the first subsection “E. purpurea extracts.....”, the first 10 lines are a description of the methodology and mostly a repetition of things already said. We agree with the reviewer on this and have significantly shortened this part. -As negative control (blank) the use of ethanol alone is more correct than spinach ethanol extracts since we cannot know if spinach extracts contain molecules with an opposite effects to that exerted by ethanol. You must exclude that ethanol may be responsible in whole or in part for the effects observed with the Echinacea extracts There is some misunderstanding here. When we said “ethanol extracts”, we meant the plant extracts were initially extracted with ethanol. These ethanol extracts were later actually dried (ethanol completely removed) and re-dissolved in DMSO, and eventually diluted by 1000 times with HEPES buffer before applied to the cells. Therefore ethanol should not contribute to the effects we observed with Echinacea. We did use 1% DMSO (dissolved in HEPES buffer) as a negative control throughout our experiments and the DMSO control did not have any effects on HEK cells. We have clarified this and added more details in the Methods section (revised manuscript, Page 6, Lines 6-8). Results -page 12 title of paragraph: the name of Thapsigargin is badly written (Thapsigarin); We thank the reviewer for pointing that out, we have corrected our spelling (revised manuscript, Page 12, Lines 1). Discussion -In view of the results with calcium free medium, to speak of “influx” of calcium is not correct, since the increase of intracellular calcium derives by the release from intracellular stores and not from the external of cells; then the term influx here as in the abstract section must be changed, for example simply with “increase”. We agree with the reviewer and have changed the term “influx” with “increase” or “elevation”. -The hypothesis of an involvement of specific receptors, such as chemokine, glutamate and purinergic receptors must be more discussed, for example explaining why a direct activation of PLC has been excluded. We agreed with the reviewer and we clarified that we have not excluded the possibility of a direct activation of PLC (revised manuscript, Page 14, Lines 13-18). -All the final part of the discussion (from page 16 line20 to page 18 line 2) is off topic We have removed 13 lines from the original discussion. Conclusion -Erase the data from literature and the last sentence. Conclude only with the data from the present study We agree with the reviewer and have edited the conclusion part as suggested. Reviewer: Ju rg Gertsch 1. In the abstract it should be stated that the compounds that induce cytosolic Ca2+ via PLC and IP3 in HEK293 cells are not yet characterized. Moreover, the finding that the calcium is induced via PLC may be important and should be stated in the abstract too. We thank the reviewer for this great suggestion. We have edited our abstract accordingly (revised manuscript, Page 3, Line 7; Page 4, Lines 8-9). 2.Why were not other cell types tested, like e.g. epithelial cells or fibroblasts? Do Kidney cells may be very particular with respect to cytosolic calcium signalling. Do the authors think that these Echinacea constituents could induce such intracellular calcium in kidney if not, what would the physiological relevance be, given that the effect is in the microM (microg/mL) range? Alkamides have previously been shown to exert functionla effects at nM concentrations (effect on cytokine regulation in whole blood), but what about the potential link of this observation in a physiological context? We have already looked at other cells (Birt et al., 2008) relating to biological activities of Echinacea. In this manuscript we want to see if Echinacea can affect a cell type that is not particularly implicated in its previously known physiological responses. We are in the process of testing other cell types. We agree this will be very interesting. We thank the reviewer for the suggestion. 3. It will be important to isolate the constituents to perform experiments and to elucidate the receptors involved in this response. We totally agree on that. We believe there are a series of related compounds in these six active fractions. Because the bioactive constituents are not one of the known compounds and present in very low concentrations, we have not yet been able to identify them. Specific points: The use of 2-APB (100 μM)is one line of evidence that IP3 is involved, but what about other approaches, e.g. heparin as an IP3 antagonist, which would be easily done. We thank the reviewer for this suggestion. We had considered testing heparin as another IP3 antagonist, however, heparin is not membrane permeative due to its high negative charge and large size. It is normally applied intracelluarly by injection (Huang et al., 1991; Salter et al., 1995) therefore will be very difficult to administrate considering the small size of the HEK cells. Since HEK293 cells do not express CB2 surface expression it is expected that SR144528 does not block this effect. Nevertheless, it is still a good control experiment. In the case of CB2 receptors, which are only targeted by certain alkamides, a link to immune processes could be established. The proposal in Fig.8 is too speculative since in this study no such cells were used. Fig. 8 is at present rather speculative and should be omitted, at least until the precise mechanisms are found. HEK293 cells are special and may not stand for physiological effects in other cell types this would have to be shown experimentally. We have revised Figure 8 and only retained the portion of the figure we have evidence for from this manuscript. Conclusions: how do the authors know whether the Ca2+ active constituents are novel or not? This can only be known once they have been isolated. That is a good point. We have changed the word “novel” to “unidentified” which is more appropriate (revised manuscript, Page 4, Line 8). Reference Birt DF, Widrlechner MP, LaLone CA, Wu L, Bae J, Solco AK, Kraus GA, Murphy PA, Wurtele ES, Leng Q, Hebert SC, Maury WJ, Price JP: Echinacea in infection. Am J Clin Nutr 2008, 87(suppl): 488S-492S Huang C-L, Takenawaj T, Ives HE. Platelet-derived growth factor-mediated Ca 2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors. The Journal of Biological Chemistry 1991, 226: 4045-4048 Salter MW, JL Hicks. ATP causes release of intracellular Ca 2+ via the phospholipase C beta/IP3 pathway in astrocytes from the dorsal spinal cord. Journal of Neuroscience 1995, 15: 2961-2971