Volume regulation in response to hypo-osmotic stress in goldfish retinal ganglion cell axons regenerating in vitro.

Goldfish retinal ganglion cell (RGC) axons regenerating in vitro were used to investigate the volume regulatory response to hypo-osmotic stress. Reducing the tonicity of the bathing medium to half strength caused an immediate swelling of axons; however, within 1 min a progressive volume reduction ensued which stabilized at near control volume over a period of 10 min. This regulatory volume decrease (RVD) was attenuated by elevated [K+]o, Ca2(+)-activated K+ channel antagonists, and calmidazolium, a potent calmodulin inhibitor. Inclusion of ATP-gamma S in the hypotonic bathing medium led to a loading of stressed axons which resulted in an excessive volume reduction that reflected an overshooting of the RVD response. The latter suggested the importance of phosphorylation/dephosphorylation reactions in the RVD response pathway. Cytochalasin D and colchicine had no effect on the development of the typical RVD response, providing no evidence of involvement of actin or microtubule cytoskeletons in the volume reduction mechanism of the immature axons. The results are consistent with the hypothesis that hypo-osmotic stress activates a calcium/calmodulin dependent membrane pathway, which probably involves transient phosphorylation, leading to a loss of cellular K+ and osmotically obligated water which restorates normal axonal volume.