Trypsin Depolarizes Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal

Round 1

Reviewer 1 Report

PARs are abundantly recognized for their roles in the development of inflammatory diseases. Trypsin is the major PAR2 cleaving protease that initiates inflammatory signaling. The role of trypsin on ICC may be involved in the regulation of inflammation of the gastrointestinal tract.  

1. Trypsin depolarized the membrane potential of ICC through the activation of NK1 and NK2. What is the mechanism? Does PAR-2 agonist depolarize the membrane potential of ICC through the activation of Neurokinin receptors?
2. What is the role of trypsin on the contraction of small intestinal smooth muscle? What is the relationship between trypsin-induced depolarization of ICC and the role of trypsin on the contraction of small intestinal smooth muscle?
3. Fig. 3 D, E, Trypsin --> AC55541

Author Response

PARs are abundantly recognized for their roles in the development of inflammatory diseases. Trypsin is the major PAR2 cleaving protease that initiates inflammatory signaling. The role of trypsin on ICC may be involved in the regulation of inflammation of the gastrointestinal tract.  

1.Trypsin depolarized the membrane potential of ICC through the activation of NK1 and NK2. What is the mechanism? Does PAR-2 agonist depolarize the membrane potential of ICC through the activation of Neurokinin receptors?

Responses) In the past studies, it can be seen that PAR2-induced small intestine contraction occurs through neurokinin receptors in the mouse small intestine [49]. Therefore, the regulation of pacemaker potential by PAR2 in ICCs may be also thought to generate neurokinin receptors. We plan to conduct further research in this area in the future.

 

Reference

49.Zhao, A.,; Shea-Donohue, T. PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors. Am. J. Physiol. Gastrointest. Liver Physiol. 2003, 285, G696-703.

 

We added this in Discussion.

2.What is the role of trypsin on the contraction of small intestinal smooth muscle? What is the relationship between trypsin-induced depolarization of ICC and the role of trypsin on the contraction of small intestinal smooth muscle?

Responses) It is known that the NK receptor is involved in trypsin-induced small intestinal smooth muscle contraction [49]. Therefore, considering the results in this paper, it may be thought that when trypsin depolarizes the pacemaker potential of ICC, this stimulation excites smooth muscle and eventually causes small intestine contraction. It can also be seen that NK receptors are involved in all these processes.

Reference

49.Zhao, A.,; Shea-Donohue, T. PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors. Am. J. Physiol. Gastrointest. Liver Physiol. 2003, 285, G696-703.

We added this in Discussion.

 

3.Fig. 3 D, E, Trypsin --> AC55541

Responses) There seems to be a misunderstanding. I think trypsin is right.

Author Response File: Author Response.docx

Reviewer 2 Report

    In this study, the authors studied the effects of trypsin on pacemaker potentials in murine small intestinal ICCs. The authors demonstrated that trypsin/PAR2 signaling regulates small intestinal motility by modulating the pacemaker activity of ICCs. The trypsin/PAR2 signaling-regulated pacemaker potential depolarization is dependent on extracellular Ca2+ and the G protein, PLC, PKC, and IP3 signaling pathways. This paper will be of interest to the researchers in this field. The overall level of the paper is well written and some important considerations are highlighted.

1. The authors should include an abstract to summarize the objective, method, result and conclusion of this study.
2. The authors were encouraged to include a schematic diagram as Figure 8 to summarize the findings by the authors.
3. In Figure 1, What is “this is a figure”?
4. The formats for the chemicals, such as CaCl2, CO2, MgCl2, Ca2+, etc. , should be changed. They should be CaCl₂, CO₂, MgCl₂, Ca²⁺.

Author Response

In this study, the authors studied the effects of trypsin on pacemaker potentials in murine small intestinal ICCs. The authors demonstrated that trypsin/PAR2 signaling regulates small intestinal motility by modulating the pacemaker activity of ICCs. The trypsin/PAR2 signaling-regulated pacemaker potential depolarization is dependent on extracellular Ca2+ and the G protein, PLC, PKC, and IP3 signaling pathways. This paper will be of interest to the researchers in this field. The overall level of the paper is well written and some important considerations are highlighted.

 

1.The authors should include an abstract to summarize the objective, method, result and conclusion of this study.

Responses) We added the Methods in Abstract. Everything else is included.

Objective: Interstitial cells of Cajal (ICCs) generate pacemaker potentials in the gastrointestinal (GI) tract. In this study, the effects of trypsin on pacemaker potentials in murine small intestinal ICCs were examined.

Method: We used the whole-cell patch-clamp analysis method.

Results: The results of whole-cell patch-clamp analysis revealed that trypsin dose-dependently depolarized pacemaker potentials and decreased their amplitude. Treatment with the antagonists of neurokinin1 (NK1) and NK2 receptors (SR-140333 and SR-48968, respectively) slightly inhibited the trypsin-induced responses. However, treatment with the combination of SR-140333 and SR-48968 completely inhibited the trypsin-induced responses. Trypsin slightly depolarized pacemaker potentials and increased their amplitude after the intracellular application of GDP-β-S. Additionally, incubation in external Ca2+-free solution inhibited trypsin-induced responses. In the presence of U-73122, staurosporine, Go6976, or xestospongin C, trypsin did not depolarize the pacemaker potentials. However, trypsin depolarized the pacemaker potentials in the presence of rottlerin. Finally, HC067047, a TRPV4 inhibitor, did not affect the trypsin-induced responses.

Conclusion: These results suggest that trypsin depolarized pacemaker potentials through NK1 and NK2 receptors in the murine small intestinal ICCs, with this effect being dependent on the G protein, phospholipase C, protein kinase C, inositol triphosphate pathways, and extracellular Ca2+ but being independent of the TRPV4 pathway. Hence, trypsin-mediated GI motility regulation must be considered for prokinetic drug development.

 

2.The authors were encouraged to include a schematic diagram as Figure 8 to summarize the findings by the authors.

Responses) We added the schematic diagram as Figure 8

 

3.In Figure 1, What is “this is a figure”?

Responses) I'm very sorry. Deleted and cleaned up.

 

4.The formats for the chemicals, such as CaCl2, CO2, MgCl2, Ca2+, etc. , should be changed. They should be CaCl₂, CO₂, MgCl₂, Ca²⁺.

Responses) We changed.

Author Response File: Author Response.docx

Reviewer 3 Report

Thank you for permitting me to review this manuscript

Please explain the lack of power calculation

Please describe with more details the patch clamp technique

Line 90-92 please provide reference

Line 167 please provide reference

 

Line 259 please provide reference

Line 291 please specify in humans  or not

Please suggest potential clinical application

 

Conclusion

Please specify in mices only

 

Author Response

Thank you for permitting me to review this manuscript

Please explain the lack of power calculation

Responses) I haven't used power calculation in patch experiments in the past 20 years. I'm not sure exactly what the question means.

 

Please describe with more details the patch clamp technique

Responses) We described with more details the patch clamp technique.

Command pulses were applied using pClamp software (version 6.1; Axon Instruments). The data were filtered at 5 kHz with Axopatch 200B amplifiers.

 

Line 90-92 please provide reference

Responses) We provided reference 23.

23.Moon, S.B.; Choi, N.R.; Kim, J.N.; Kwon, M.J.; Kim B.S.; Ha, K.T.; Lim, E.Y.; Kim, Y.T.; Kim, B.J. Effects of black garlic on the pacemaker potentials of interstitial cells of Cajal in murine small intestine in vitro and on gastrointestinal motility in vivo. Anim. Cells. Syst. (Seoul) 2022, 26, 37-44.

 

Line 167 please provide reference

Responses) We provided reference 36.

36.Boichot, E.; Germain, N.; Emonds-Alt, X.; Advenier, C.; Lagente, V. Effects of SR 140333 and SR 48968 on antigen and substance P-induced activation of guinea-pig alveolar macrophages. Clin. Exp. Allergy 1998, 28, 1299-1305.

 

Line 259 please provide reference

Responses) We already provided reference 26,27,28, and 32.

26.Lewis, G.; Christiansen, L.; McKenzie, J.; Luo, M.; Pasackow, E.; Smurnyy, Y.; Harrington, S.; Gregory, P.; Veres, G.; Negre, O.; Bonner, M. Staurosporine Increases Lentiviral Vector Transduction Efficiency of Human Hematopoietic Stem and Progenitor Cells. Mol. Ther. Methods Clin. Dev. 2018, 9, 313-322.

27.Grandage, V.L.; Everington, T.; Linch, D.C.; Khwaja, A. Gö6976 is a potent inhibitor of the JAK 2 and FLT3 tyrosine kinases with significant activity in primary acute myeloid leukaemia cells. Br. J. Haematol. 2006, 135, 303-316.

28.Gschwendt, M.; Müller, H,J,; Kielbassa, K.; Zang, R.; Kittstein, W.; Rincke, G.; Marks, F. Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 1994, 199, 93-98.

32.Saleem, H.; Tovey, S.C.; Molinski, T.F.; Taylor, C.W. Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor. Br. J. Pharmacol. 2014, 171, 3298-3312.

 

Line 291 please specify in humans or not

Responses) We specified.

 

Please suggest potential clinical application

Responses) We suggested potential clinical application. 

In general, trypsin is a digestive enzyme that's critical for good health. In addition, the results of this paper indicate that trypsin may play a role in regulating gastrointestinal motility. Therefore, it is considered as one of the good proteases that can treat abnormalities of the digestive system by controlling the concentration of trypsin.

 

Conclusion

Please specify in mices only

Responses) We specified.

Author Response File: Author Response.docx