|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Cholinergic effects on KC directional migration. (A) Chemotactic effects of cholinergic agonists on human KCs. Second-passage foreskin KCs were loaded into the chemotaxis AGKOS plates and incubated overnight to allow cells to settle, after which increasing concentrations of the agonists nicotine (Nic), epibatidine (Epi) and choline (Cln) diluted in PBS vs PBS alone (control; C) were added to the chemoattractant well (Fig. 1B). The agonist concentrations are shown on the ordinate axis as 10–x M. The plates were incubated for 10 days with daily refreshment of the chemoattractant solution. A statistically significant (P<0.05) increase in the directional migration distance (DMD) was observed starting at 10 pM nicotine, 1 pM epibatidine and 0.1 mM choline. The DMD of control and experimental cells are expressed as means±s.d. µm. An asterisk denotes statistical significance, P<0.05, compared with control. (B) Subtype-selective antagonists of nAChRs exhibit differential inhibitory effects on directional migration of KCs. The chemotaxis of human KCs was elicited using the most efficient concentration of each agonist shown in A. The antagonists 50 µM mecamylamine (Mec), 1 µM 
-bungarotoxin (BTX) and 5 µM strychnine (Str) were added directly to the KC well (Fig. 1B), being dissolved in KGM that was changed daily. The DMD values are expressed as means±s.d. µm. An asterisk denotes statistical significance (P<0.05) compared with DMD of the control KCs that were not exposed to antagonists. Significant differences between DMD values of KCs treated with Mec vs BTX are indicated by arrows at the top. (C) Decreased directional migration of human KCs with silenced 7 nAChR. Second-passage human KCs were loaded into the chemotaxis AGKOS plates, incubated for 18 hours to allow cells to adhere to the dish bottom and transfected with COs or anti-nAChR subunit AsOs, after which 1 mM choline was added to the chemoattractant well (Fig. 1B) and the incubation was continued for 10 days. The results are expressed as means±s.d. of control. (D) Decreased directional migration of murine KCs from 7 knockout mice. Second-passage KCs grown from the epidermis of at least three neonatal 3–/–, 5–/–, 7–/–, 9–/–, ß2–/– or ß4–/– mice, or their +/+ littermates were loaded into the chemotaxis AGKOS plates and allowed to migrate towards the concentration gradient of choline for 10 days, as described in C. An asterisk denotes statistical significance (P<0.05) compared with the DMD of 7+/+ KCs. (E) Effects of the nAChR signaling modifiers on directional migration of human KCs. The chemokinetic response to choline was measured in the chemotaxis AGKOS plates using HC-3 (20 µM) treated KCs in which endogenous ACh was substituted by exogenously added CCh (1 mM) (as in Fig. 2F). The cells were fed with KGM containing 10 µM BAPTA/AM, 10 µM KN-62 or KN-93, 1 µM chelerythrine (Chlrn), 1 µM Gö-6976, 5 µM rottlerin (Rtln), 100 pg ml–1 toxin B (TxB), 10 µg ml–1 C3 exoenzyme (C3), 5 µM Y-27632, 100 nM wortmannin (Wtmn) or 10 µM Ly-294002. The results are expressed as means±s.d. of untreated control, taken as 100%. Asterisks indicate significant (P<0.05) differences from control.
|
