Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder
Abstract
:1. Introduction
2. Results
2.1. Spontaneous Ca2+ Activity in Cultured Human suICs
2.2. Single Cell Mechanical Stimulation by Glass Micropipette
2.3. Intercellular Propagation of the Ca2+ Signal
2.4. Shear Stress Evoked Calcium Transients
2.5. Hypotonic Stimulation
2.6. Immunocytochemical Characterization of Cultured suICs
3. Discussion
- (ii) Mechanical stimulation of human suICs either by direct indentation of the plasma membrane, swelling of the cells by hypotonic Ringer solution or membrane deformation by sheer stress induced calcium transients. Interestingly, calcium transients were also elicited when releasing the sheer stress by reduction of the flow rate to normal flow. This indicates that the cells are also capable of sensing the changing of the pressure in addition to the force of the pressure.
- (iii) Calcium signals propagated not only intracellularly but were transmitted to neighboring cells. This indicates that the suICs formed a functional syncytium, as previously shown [21,22]. Interestingly, we observed two populations of neighboring cells, with approximately 87% of the cells showing a delay of calcium activation after single cell stimulation, resulting in an intercellular signal propagation velocity of about 20 µm/s, while in 20 of 152 cells (13%), the delay was much shorter, with a calculated propagation speed of ≥ 50 µm/s. These cells were similarly found in close proximity to the stimulated cell (<50 µm radius), as well as within a distance of 50 to 250 µm (Figure 7D). We hypothesized that the latter cells were directly connected to the stimulated cell via long thin cell processes, thus resembling telocytes [36], which also have been proposed to be present in the upper lamina propria (ULP) of the human bladder [26,37]. This idea was directly supported by dye coupling experiments, regularly demonstrating cells with long cytoplasmic processes in cultured suICs (Figure 12), and our immunocytochemical experiments indicating the presence of typical type 1 telocytes (PDGFRa+/CALR+/aSMA−) and of PDGFRa+/CALR+/aSMA+ hybrid (type 2) telocytes (Figure 10, Figure 11, Figure S2). We found type 1 telocytes, which were characterized by very small nuclei and small cell bodies showing long thin bipolar cell protrusions spanning up to 200 µm. These cells were observed at a rate of 12.5% in early cell cultures and decreased to around 7% with increasing confluency. Since we used the cell cultures at ≥ 80% confluency (e.g., Figure 4), it is most probable that we did not directly stimulate these cells. However, the small fraction of typical telocytes would well correspond to the observed 13% of cells with short Ca2+ signal delay in up to 250 µm distance (Figure 7D), thus promoting a fast calcium wave distribution over the cellular syncytium. Interestingly, we found no Cx43-IR in typical myofibroblasts accounting for less than 1% of the cells. These very large cells, therefore, would not be considered to contribute to the propagation of the calcium signal.
4. Materials and Methods
4.1. Ethical Statement
4.2. Cell Cultures
4.3. Chemicals and Solutions
4.4. Calcium Imaging
4.5. Data Analysis and Statistics
4.6. Stimulation Experiments
4.6.1. Single Cell Mechanical Stimulation
4.6.2. Mechanical Stimulation by Shear Stress
4.6.3. Hypotonic Stimulation
4.7. Dye-Coupling Experiments
4.8. Confocal Imaging
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AUC | area under the curve |
aSMA | alpha-smooth muscle actin |
BOO | bladder outlet obstruction |
CALR | calreticulin |
Cx43 | connexin 43 |
DAPI | 4′,6-diamidin-2-phenylindol |
FITC | fluorescein isothiocyanate |
fps | frames per second |
fura-2AM | fura-2-acetoxymethylester |
IC/BPS | interstitial cystitis/bladder pain syndrome |
OAB | overactive bladder |
PDGFRa | platelet derived growth factor receptor-alpha |
SD | standard deviation |
SEM | standard error of the mean |
suIC | suburothelial interstitial cells |
TRP | transient receptor potential |
VIM | vimentin |
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Group | Mean | SD | N |
---|---|---|---|
1 | 56.5466 | 27.46916 | 1913 |
2 | 221.3514 | 70.75953 | 582 |
3 | 488.9613 | 72.17626 | 245 |
4 | 743.6639 | 69.71185 | 134 |
5 | 978.3084 | 66.73132 | 70 |
6 | 1239.981 | 66.15836 | 29 |
Cell Group | 0 | 50 | 100 | 150 | 200 | 250 | Control |
---|---|---|---|---|---|---|---|
Distance (µm) | Stimulated | <= 50 | >50 ≤ 100 | >100 ≤150 | >150 ≤ 200 | >200≤ 250 | NA |
Number of cells | 14 | 81 | 155 | 213 | 139 | 16 | 619 |
Active cells (n) | 14 | 44 | 77 | 67 | 41 | 8 | 34 |
Mean (%) | 100 | 54.3 | 49.7 | 31.5 | 29.5 | 50 | 5.5 |
95% CI (%) | 43.2–65.4 | 40.3–50.2 | 25.2–37.7 | 21.8–37.2 | 12.9–51.6 | 3.7–7.3 | |
Peak ampl. mean | 2242 | 1025 | 954 | 887 | 848 | 1090 | 599 |
95% CI | 1683–2800 | 831–1219 | 840–1068 | 766–1008 | 639–1058 | 363–1817 | 489–711 |
Peak AUC (au) | 476737 | 189641 | 175652 | 157005 | 115,647 | 97981 | 41535 |
95% CI | 228,359–725,115 | 119,516–259,766 | 139,339–211,966 | 122,889–191,121 | 80,430–150,865 | 9981–185,982 | 28,387–54,684 |
Peak duration (s) | 40.9 | 36.8 | 36.8 | 33.4 | 28.1 | 39.3 | 28.3 |
95% CI | 29.4–52.6 | 32.8–40.9 | 34.1–39.6 | 30.5–36.3 | 24.6–31.7 | 19.4–39.2 | 23.4–33.2 |
Peak delay (s) | NA | 6.534 | 11.65 | 18.34 | 18.1 | 15.68 | NA |
95% CI | 4.1–8.9 | 9.4–13.9 | 15.4–21.3 | 12.9–23.3 | 10.1–21.3 |
Peak | Section A | Section B | Section C | p–Value |
---|---|---|---|---|
Frequency (min−1) | 0.62 (522) | 0.70 (554) | 0.75 (559) | A/B: <0.0001 § |
[0.59–0.64] | [0.68–0.73] | [0.72–0.78] | B/C: 0.0039 § | |
Amplitude (FI) | 269.4 (1345) | 344.7 (1628) | 207.6 (1757) | A/B: <0.0001 $ |
[245.6–293.19] | [319.4–370.0] | [192.1–223.2] | B/C: <0.0001 $ | |
Duration (s) | 34.26 (1345) | 39.43 (1628) | 35.74 (1757) | A/B: <0.0001 $ |
[32.99–35.54] | [38.03–40.83] | [34.59–36.90] | B/C: <0.0001 $ | |
AUC | 5789 (1345) | 8860 (1628) | 4602 (1757) | A/B: <0.0001 § |
[5149–6430] | [8060–9660] | [4172–5032] | B/C: <0.0001 § |
Osmotic Conc. | 309 | 232 | 154 | p–Value |
---|---|---|---|---|
(mOsm/L) | Control | Hypo25 | Hypo50 | |
frequency (min–1) | 0.02 (44) | 0.45 (36) | 0.35(58) | Hypo25/Hypo50 |
[0.0–0.06] | [0.39–0.49] | [0.34–0.36] | <0.0001 $ | |
amplitude (FI) | 32.63 (1) | 733.2 (47) | 2147 (59) | Hypo25/Hypo50 |
[NA] | [579.8–886.7] | [1891–2403] | <0.0001 $ | |
duration (s) | 21 (1) | 93 (47) | 114.6 (59) | Hypo25/Hypo50 |
[NA] | [80.19–105.8] | [109.4–119.8] | 0.0021 $ | |
AUC | 419.6 (1) | 37,707 (47) | 119,702 (59) | Hypo25/Hypo50 |
[NA] | [27,896–47,517] | [102,882–136,421] | <0.0001 $ |
Target | Host Isotype | Class | Conjugate | Dilution | Product Number | Source |
---|---|---|---|---|---|---|
vimentin (VIM) | mouse gG1 | Monoclonal | na | 1:200 | V6389 | a |
alpha-smooth muscle actin (aSMA) | mouse IgG2a | Monoclonal | na | 1:1000 | A2547 | a |
platelet-derived growth factor receptor-alpha (PDGFRa) | goat | Polyclonal | na | 1:100 | AF-307-NA | b |
calreticulin (CALR) | rabbit | Polyclonal | na | 1:600 | ab2907 | c |
connexin 43 (Cx43) | rabbit | Polyclonal | na | 1:500 | C6219 | a |
mouse IgG1 | goatIgG | Polyclonal | Alexa Fluor® 488 | 1:500 | A21121 | d |
mouse IgG2a | goat IgG | Polyclonal | Alexa Fluor® 633 | 1:500 | A21136 | d |
rabbit IgG | goat IgG (H+L) | polyclonal | Alexa Fluor® 555 | 1:500 | A21121 | d |
mouse IgG | donkey IgG (H+L) | monoclonal | Alexa Fluor® 488 | 1:500 | A21202 | d |
goat IgG | donkey IgG | polyclonal | Alexa Fluor® 555 | 1:500 | A41232 | d |
rabbit IgG | donkey IgG | polyclonal | Dylight® 649 | 1:500 | 711-495-152 | e |
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Neuhaus, J.; Gonsior, A.; Cheng, S.; Stolzenburg, J.-U.; Berger, F.P. Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder. Int. J. Mol. Sci. 2020, 21, 5474. https://doi.org/10.3390/ijms21155474
Neuhaus J, Gonsior A, Cheng S, Stolzenburg J-U, Berger FP. Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder. International Journal of Molecular Sciences. 2020; 21(15):5474. https://doi.org/10.3390/ijms21155474
Chicago/Turabian StyleNeuhaus, Jochen, Andreas Gonsior, Sheng Cheng, Jens-Uwe Stolzenburg, and Frank Peter Berger. 2020. "Mechanosensitivity Is a Characteristic Feature of Cultured Suburothelial Interstitial Cells of the Human Bladder" International Journal of Molecular Sciences 21, no. 15: 5474. https://doi.org/10.3390/ijms21155474