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Figures
- Fig. 1.
Scratching monolayers stimulates cAMP synthesis in MDCK cells. Confluent monolayers of MDCK cells were maintained in 1.8 mM Ca2+ Ringer's solution containing 100 µM IBMX, a phosphodiesterase inhibitor. Cells were then wounded by scratching monolayers with a 27G needle 20 times, or treated with 100 µM ATP or 100 µM forskolin. The amount of cAMP was quantified by immunoassay and normalized to the amount of total protein. The number of experiments is indicated in parentheses. *P=0.0006.
- Fig. 2.
Cell membrane disruption stimulates cAMP production in neighboring cells. (A) ‘w’ in the fluorescence image of MDCK cells expressing Green Upward cADDis indicates a wounded cell. Cells adjacent to the wounded cell were labeled with numbers in order of their proximity to the wounded cell. A cell was wounded at time zero with a glass needle in 1.8 mM Ca2+ Ringer's solution, and the time course of changes in fluorescence intensity of cADDis was plotted for neighboring cells (1–3). The image shown in this figure was acquired 90 s after cell membrane disruption. See also Movie 1. (B) Cells were wounded at time zero with a glass needle in the absence or presence of 20 U/ml apyrase, and changes in fluorescence intensity in neighboring cells were compared. The number of observed cells is indicated in parentheses. P=0.0007 (a–a′); P=0.0427 (b–b′); P=0.0197 (c–c′).
- Fig. 3.
ATP and forskolin stimulate cAMP synthesis in MDCK cells. Cells expressing Green Upward cADDis were treated with either 100 µM ATP or 100 µM forskolin at the time indicated by arrows, and the changes in fluorescence intensity of cADDis were recorded. The arrowhead indicates the transient decrease in fluorescence intensity. The number of observed cells is indicated in parentheses.
- Fig. 4.
Cell membrane disruption induces Ca2+ mobilization in neighboring MDCK cells. (A) Cells loaded with Calcium Green-1 AM were wounded at time zero with a glass needle in the presence or absence of extracellular Ca2+, and changes in fluorescence intensity in the cytoplasmic region were compared. Cells were numbered as per Fig. 2A. The number of observed cells is indicated in parentheses. See also Movie 2. (B) To compare the initial phase of increase in [Ca2+]i, data from cell #1 in A were expanded. *P=0.0333; NS, not significant.
- Fig. 5.
Cell membrane disruption potentiates membrane resealing in neighboring cells in a PKA- and PKC-dependent manner. Cells loaded with Calcein Red-Orange AM were initially wounded with a glass needle, and changes in fluorescence intensity of Calcein Red-Orange were monitored. Neighboring cells were wounded 5 min later and changes in fluorescence intensity were recorded. The resealing rate was defined as the reciprocal of resealing time in seconds. For cells that failed to reseal, the rate was defined as zero. The number of observed cells is indicated in parentheses. *P=0.002.
- Fig. 6.
Potentiation of membrane resealing induced by ATP is suppressed by PKA and PKC inhibitors in MDCK cells. Cells were pretreated for 10 min with either kinase inhibitors or DMSO. Then, cells were wounded with a glass needle after the addition of 100 µM ATP in the presence or absence of inhibitors, respectively, and the resulting resealing rates were analyzed. As a control, cells treated with 100 µM AMP were wounded with a glass needle. Resealing rates were analyzed 5–20 min after the addition of nucleotides. The number of observed cells is indicated in parentheses. *P=0.0007; †P<0.0001.
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