To study the role of β2-adrenergic receptors and intracellular cAMP (cyclic AMP) in the electric field-mediated directional migration of keratinocytes that might play an important part in initiating re-epithelialization for wound healing.
Re-epithelialization of wounded skin is necessary for wound closure and requires directional keratinocyte migration towards the center of the wound. The electric field generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many β2-adrenergic receptors, but their role in the epidermis is unknown. The authors have previously shown that beta-adrenergic receptors agonists decrease keratinocyte migration in a cAMP independent mechanism involving the activation of protein phosphatase 2A.
|Exposure duration||continuous for 1 h|
|Additional info||Reference article: Pullar, C. E., Isseroff, R. R. and Nuccitelli, R. (2001). Cyclic AMP dependent protein kinase A plays a role in the directed migration of human keratinocytes in a DC electric field. Cell Motil. Cytoskeleton 50, 207-217.|
|Chamber||Galvanotaxis chamber maintained at 36°C|
|Additional info||Galvanotaxis chamber is composed of a rectangular Plexiglass frame with two media reservoirs on opposite sides to which a 45 x 50 mm glass coverslip is attached to form the chamber bottom. The keratinocytes are plated onto the collagen-coated center of the chamber between two coverslip spacers (25 x 10 mm). A third 25 mm² coverslip is placed on top and rests approx. 100-105 µm above the center panel and is sealed on top of the spacer coverslips with silicone high-vacuum grease.|
|electric field strength||1 V/cm||-||measured||-||-|
When keratinocytes were exposed to higher concentrations of beta-adrenergic receptor agonist (0.1 µM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV/mm electric field. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost - essentially, keratinocytes were 'blinded' to the directional cue.
Preincubating the keratinocytes with β-antagonist restored directional migration, demonstrating that the 'blindness' was β2-adrenergic receptor mediated.
Incubation of cells with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar 'blinding' to the electric field, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration. However, rp-cAMP pretreatment before β-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration.
This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. beta-adrenergic receptor agonists and antagonists could be valuable tools for modulating re-epithelialization. Thus, beta-adrenergic receptors regulate the two distinct components of keratinocyte directional migration differently: migration speed via a cAMP-independent mechanism and galvanotaxis by a cAMP-dependent one.