Some Factors Affecting Sodium Outflux in Internally Dialyzed Squid Axons

Although ATP has been shown to be an energy source for sodium extrusion in injected squid axons (CaldweU, Hodgkin, Keynes, and Shaw, 1960) as well as dialyzed axons (Brinley and Mullins, 1967; Mullins and Brinley, 1967), the ability of other high-energy phosphate compounds to support sodium extrusion has not been thoroughly studied. An internal dialysis technique has been used for such a study because it allows the experimenter not onlytoadd diffusible substrates, but also to remove them from the axoplasm. The ability to remove unwanted materials is an important experimental consideration because some of the possible fuel sources studied, e.g. phosphoarginine (PA) and phosphoenolpyruvate (PEP), are known to participate in phosphoryl transferase reactions in squid axoplasm: thus PA qADP = A dATP. Therefore the addition of such substrates to axoplasm containing even small amounts of ADP could be expected to generate ATP by phosphorylating ADP. Any effect of such compounds upon sodium outflux could therefore be ascribed just as well to ATP as to the high-energy compound itself. It would appear that a critical test of the ability of a high-energy phosphate compound to fuel sodium extrusion must be performed upon axoplasm essentially free of substrates capable of phosphorylation, particularly ADP and AMP. The basic apparatus and perfusing solutions used in the experiments to be presented have already been described (Brinley and Mullins, 1966, 1967; Mullins and Brinley, 1967; MuUins, 1967; Mullins, 1968). The manner in which the porous glass capillary was used to prepare a length of axoplasm free of ADP and AMP has been described (Mullins and Brinley, 1967). Subsequent dialysis with a test compound occurs within a region essentially free of ADP-AMP. Since the level of ADP-AMP to be expected in the effluent fluid during such a dialysis is well below the detectable limits of conventional chromatographic or enzymatic assay techniques, a direct measurement of the residual ADPAMP was not possible. However, an indirect measure of the effectiveness of the predialysis was obtained by measuring the ATP levels in the effluent dialysis fluid first in the absence and then in the presence of the phosphorylating agent. The effectiveness of the "predialysis" procedure can be judged from the fact that when both ends of