In the absence of streptomycin, minoxidil potentiates the mitogenic effects of fetal calf serum, insulin-like growth factor 1, and platelet-derived growth factor on NIH 3T3 fibroblasts in a K+ channel-dependent fashion.

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

An in vitro model for the study of human hair growth.

Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermosubcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watch makers' forceps. Isolated hair follicles maintained free floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinized hair shaft, and was not associated with the loss of hair follicle morphology. [Methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labeling of keratins showed that their patterns of synthesis did not change with maintenance. Serum was found to inhibit hair follicle growth in vitro; and when follicles were maintained in serum-free medium, they grew for up to 10 days, suggesting that in vitro the hair follicles are able to regulate their own growth, possibly by the production of relevant growth factors. This may prove useful in identifying the autocrine/paracrine mechanisms that operate in the hair follicle. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth regulatory effect on hair follicles in vitro and that EGF and its other receptor ligand TGF-alpha mimic the in vivo depilatory effects of EGF that have been reported for sheep and mice.