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First published online 19 April 2005
doi: 10.1242/jcs.02330

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Cyclic AMP mediates keratinocyte directional migration in an electric field

Department of Dermatology, University of California, Davis, CA 95616, USA

View larger version (21K): [in a new window]   Fig. 1. Human keratinocytes migrate towards the cathode in the presence of 100 mV mm–1 DC EFs. Images of the migration paths were captured every 10 minutes and the translocation distance and directionality calculated. For each experiment, the data were plotted using a circle graph. Each cells position at time (t)=0 minutes is at the origin (0,0) and its final position at the end of the 1 hour exposure to the DC EF is plotted as a single point on the graph. The radius of each circle represents 120 µm of translocation distance. The cathode is at the top of each graph (0°) and the anode at the bottom (180°). (A) Keratinocytes in the absence of an EF are the negative control (n=153). (B) Keratinocytes in the presence of an EF are the positive control (n=132). The average track cosine for each 10 minute time period was plotted against time for keratinocytes in the absence of an EF (negative control, C) or the presence of an EF (positive control, D). Individual cell tracks are displayed to show random migration (Non-Field) (E) versus EF-mediated directional migration (Field) (F). Bar, 25 µm. The migration rate and the cosine of the migration angle [cos()] for the keratinocytes were measured after 1 hour in the absence (Non-Field) or presence (Field) of an applied DC EF (G). Solid bars (left) represent migration rate, striped bars (right) represent directionality [net cos()]. Error bars indicate s.e.m. *P<0.01. The data shown are combined from three independent experiments on two separate cell strains.

View larger version (28K): [in a new window]   Fig. 2. ß-Adrenergic agonists attenuate keratinocyte galvanotaxis at low concentrations but have no effect on migration rate. Circle graphs (radius 120 µm) were plotted to represent the translocation of keratinocytes after 1 hour in an applied DC EF of 100 mV mm–1 in the presence of increasing concentrations of ß-adrenergic agonist (A). The average track cosine for each 10 minute time period was plotted against time for untreated and 0.1 nM ß-adrenergic-agonist-treated cells in the presence of an EF (B). The migration rate and cosine of the migration angle [cos()] for control and ß-adrenergic-agonist-treated cells (0.1 pM, 0.1 nM, 0.1 µM) were measured after 1 hour in the presence of an applied DC EF. Solid bars (left) represent migration rate, striped bars (right) represent directionality [cos()]. Field control, n=132; 0.1 pM ß-agonist, n=100; 0.1 nM ß-agonist, n=137; 0.1 µM ß-agonist, n=66. The data shown are combined from three independent experiments on two separate cell strains. Error bars indicate s.e.m. *P<0.01.

View larger version (26K): [in a new window]   Fig. 3. ß-Adrenergic antagonists prevent the ß-adrenergic-agonist-mediated attenuation of keratinocyte galvanotaxis. The migration rate and cosine of the migration angle [cos()] for untreated, ß-adrenergic-agonist-treated and ß-adrenergic-antagonist-treated cells, and cells pretreated with antagonist before agonist addition were measured after 1 hour in the presence of an applied DC EF. Solid bars (left) represent migration rate, striped bars (right) represent directionality [cos()] (A). Field control, n=132; 0.1 nM ß-agonist, n=137; 20 µM timolol, n=69; ß-antagonist/ß-agonist, n=92. The data shown are combined from three independent experiments on two separate cell strains. Error bars indicate s.e.m. *P<0.01. Circle graphs (radius 120 µm) were plotted to represent the translocation of keratinocytes after 1 hour in an applied DC EF of 100 mV mm–1 in the presence of 0.1 nM ß-adrenergic agonist alone or cells pretreated with 20 µM antagonist before agonist addition at time 0 (B). The average track cosine for each 10 minute time period was plotted against time for untreated and 20 µM ß-adrenergic-antagonist-treated cells in the presence of an EF (C).

View larger version (29K): [in a new window]   Fig. 4. The active cAMP analog sp-cAMP also attenuates keratinocyte galvanotaxis. The migration rate and cosine of the migration angle [cos()] for control, ß-adrenergic-agonist-treated (0.1 nM), sp-cAMP-treated cells (50 µM) and cells pretreated with sp-cAMP before agonist addition were measured after 1 hour in the presence of an applied DC EF. Solid bars (left) represent migration rate, striped bars (right) represent directionality [cos()]. Field control, n=132; 0.1 nM ß-agonist, n=137; 50 µM sp-cAMP, n=67; sp-cAMP/0.1 nM ß-agonist, n=93. The data shown are combined from three independent experiments on two separate cell strains. Error bars indicate s.e.m. *P<0.01 (A). Circle graphs (radius 120 µm) were plotted to represent the translocation of keratinocytes after 1 hour in an applied DC EF of 100 mV mm–1 in the presence of 0.1 nM ß-adrenergic agonist alone or cells treated with 50 µM sp-cAMP before agonist addition at time 0 (B). The migration rate and cosine of the migration angle [cos()] for control, PTX-treated cells (100 ng ml–1) and cells pretreated with PTX before agonist addition were measured after 1 hour in the presence of an applied DC EF. Solid bars (left) represent migration rate, striped bars (right) represent directionality [cos()]. Field control, n=132; 100 ng ml–1 PTX, n=67; 50 µM PTX/0.1 nM ß-agonist, n=96. The data shown are combined from three independent experiments on two separate cell strains. Error bars indicate s.e.m. *P<0.01 (C).

View larger version (31K): [in a new window]   Fig. 5. The inactive cAMP analog rp-cAMP prevents the ß-adrenergic-agonist-mediated attenuation of keratinocyte galvanotaxis. Circle graphs (radius 120 µm) were plotted to represent the translocation of keratinocytes after 1 hour in an applied DC EF of 100 mV mm–1 in the presence of ß-adrenergic agonist alone (0.1 nM), rp-cAMP alone (50 µM) or cells pretreated with rp-cAMP before ß-adrenergic-agonist addition (A). The migration rate and cosine of the migration angle [cos()] for untreated, ß-adrenergic-agonist-treated (0.1 nM), rp-cAMP-treated (50 µM) cells or cells pretreated with rp-cAMP (50 µM) before ß-adrenergic-agonist (0.1 nM) addition were measured after 1 hour in the presence of an applied DC EF. Solid bars (left) represent migration rate, striped bars (right) represent directionality [cos()]. Field control, n=132; 0.1 nM ß-agonist, n=137; 50 µM rp-cAMP, n=61; rp-cAMP/0.1 nM ß-agonist, n=72. The data shown are combined from three independent experiments on two separate cell strains. Error bars indicate s.e.m. *P<0.01 (B). The average track cosine for each 10 minute time period was plotted against time for rp-cAMP-treated cells and cells pretreated with rp-cAMP before 0.1 nM ß-adrenergic-agonist addition in the presence of an EF (C).

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