Osmometrically determined characteristics of the cell membrane of squid and lobster giant axons

Lobster and squid giant nerve fibers respond differently when subjected to osmotic challenges. The axons proper, as distinct from the total (fiber) complex formed by the axon and connective sheath, both behave as "fast" osmometers for changes in the concentration of NaCl, but the maximum degree of swelling in hyposmotic media is by about 60% in lobster and only by 20% in squid. The relative volume intercepts of the van't Hoff relation are about 0.2 for lobster and 0.4 for squid. The sheaths of both axons undergo only small, apparently passive changes in volume. Lobster axons are permeable to Cl, but squid axons are impermeable to this anion. Lobster axons are also permeable to glycerol. The implications of the data as to the nature of volume regulation of cells are discussed.     

Reexamination of the double sucrose gap technique for the study of lobster giant axons. Theory and experiments

The double sucrose gap technique for the study of lobster giant axons has been reexamined. The leakage behavior of the system cannot be successfully modeled by conventional sucrose gap theory, but is accounted for by the McGuigan-Tsien model that takes into account the cable properties of membrane under sucrose. The facts of high-leakage conductance and the ability to maintain large resting potentials in the face of low sucrose gap resistance lead to a hypothesis that membrane resistance under sucrose is very low because of a large negative surface potential. Computer simulations of the leakage behavior of the conventional gap model and the McGuigan-Tsien model were compared with experimental measurements on lobster axons using normal sucrose or sucrose doped with Na+, Ca2+ or La3+ ions. As the concentration of doping ion increased, the leakage rose, but the species of doping ion had more influence on leakage than gap resistance. At equal gap resistance, leakage decreased with an increase in valence of the doping species. Leakage was even lower in La-doped sucrose at 20 M omega gap resistance than in normal sucrose at 200 M omega gap resistance. Resting potentials decreased with decreasing gap resistance and increasing valence of the doping species. Resting potential behavior was successfully simulated with a hybrid model consisting of a point node flanked by infinite cables and a shunt between ground and the voltage-measuring pool. The data support the hypothesis that the membrane resistance under sucrose is low and that it can be raised by doping the sucrose with multivalent cations, with La3+ being particularly effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium efflux in Myxicola giant axons

Several properties of the Na pump in giant axons from the marine annelid Myxicola infundibulum have been determined in an attempt to characterize this preparation for membrane transport studies. Both NaO and KO activated the Na pump of normal microinjected Myxicola axons. In this preparation, the KO activation was less and the NaO activation much greater than that found in the squid giant axon. However, when the intracellular ATP:ADP ratio of the Myxicola axon was elevated by injection of an extraneous phosphagen system, the K sensitivity of Na efflux increased to the magnitude characteristic of squid axons and the activating effect of NaO disappeared. Several axons were injected with Na2SO4 in order to determine the effect of elevated Nai on the Na efflux. Increasing Nai enhanced a component of Na efflux which was insensitive to ouabain and dependent on [Ca] in Na-free (Li) seawater. After subtracting the CaO-dependent fraction, Na efflux was related linearly to [Na]i in all solutions except in K-free (Li) seawater, where it appeared to reach saturation at high [Na]i.     

Analysis of potassium conductance in squid giant axons

Assuming a model of facilitated ionic transport across axonal membranes proposed by McIlroy (1975) and extended by McIlroy and Hahn (1978), it is shown that if the selectivity coefficient, π_K, of the potassium conducting system ?59 the permeabilityP _Ks, of the periaxonal barrier of the squid giant axon for K⁺ ions?(1.2±0.44)×10^?4 cm sec^?1 and the thickness of the periaxonal space ?477±168 Å. Using a value (10^?4 cm sec^?1) ofP _Ks in the foregoing range the experimental curves for the steady state membrane ionic conductance versus measured membrane potential difference (p.d.), ?, of Gilbert and Ehrenstein (1969) are corrected for the effect of accumulation of K⁺ in the periaxonal space. This correction is most marked for the axon immersed in a natural ionic environment, whose conductance curve is shifted ?70mV along the voltage axis in the hyperpolarization direction. By assuming that the physico-chemical connection between a depolarization of the axonal membrane and the consequent membrane conductance changes is a Wien dissociative effect of the membrane's electric field on a weak electrolyte situated in the axolemma, the position of the peaks of the corrected conductance versus ? curves can be identified with zero membrane electric field and hence with zero p.d.across the axolemma. A set of values for the double-layer p.d.s at the axonal membrane interfaces with the external electrolytes in the vicinity of the K⁺ conducting pores can therefore be deduced for the various external electrolytes employed by Gilbert and Ehrenstein. A model of these double-layer p.d.s in which the membrane interfaces are assumed to possess fixed monovalent negatively charged sites, at least in the neighbourhood of the K⁺ conducting pores, is constructed. It is shown that, using the previously deduced values for the doublelayer p.d.s, such a model has a consistent, physically realistic solution for the distance between the fixed charged sites and for the dissociation constants of these sites in their interaction with the ions of the extramembrane electrolytes.     

Magnesium content and net fluxes in squid giant axons

The Mg content of axons freshly dissected from living specimens of the tropical squid Doryteuthis plei was determined by atomic absorption spectroscopy to be 4.2 +/- 0.2 mmol/kg axoplasm. The axon's ability to maintain this physiological content of total intracellular Mg([Mg]i) was studied. Mgi was shown to be a linear function of Mgo when Mgo of incubating fluid was varied between 0 and 250 mM. When Mgo = 15 mM, Mgi was found to be the same in incubated fibers as in fibers freshly dissected. Mgi levels were unaffected by depolarization of the membrane by high Ko. Stimulation resulted in an extra influx of Mg of 0.05 pmol/(cm2 . impulse) when Mgo = 55 mM. Mgi was found to be a complicated function of the concentration of extracellular Na or Li (Xo), which was substituted for Tris. With 385 mM Lio the Mgi level was found to be 2.5-fold larger than the level observed with 385 mM Nao after incubation for 3 h. The function relating Mgo to Xo was qualitatively unaffected in axons poisoned with the mitochondrial uncoupler carbonyl cyanide, p-trifluorome-thoxy-phenylhydrazone (FCCP) and the inhibitor of glycolysis, iodoacetic acid (IAA); the absolute levels of Mgi, however, were some 30% higher in the poisoned axons at all [X]o explored. 2 h incubation of axons in a 333 mM Mg, 40 mM Li solution increased Mgi 3.5-fold in control axons and 5-fold in poisoned axons. These Mg-loaded axons were able to recover physiological levels of Mgi with a half-time of 3-5 h only if kept in a solution which contained Na (220 mM) regardless of whether the axons had been inhibited with FCCP + IAA. Therefore, it may be concluded that the physiological Mgi concentration can be maintained by the Na electrochemical gradient, even when the axon is metabolically poisoned.     

Temporally coherent organization and instabilities in squid giant axons

Instabilities and dynamic structure of the modified Hodgkin-Huxley equations (Adelman & FitzHugh, 1975) for sensitized axons were studied as a function of the sodium concentration in the external medium surrounding the axon. At the same time electrophysiological activities in squid giant axons were experimentally observed to confirm the results of the numerical calculation. It was found that the resting state of the axon was thermodynamically equivalent to a thermodynamic structure of an asymptotically stable equilibrium point. The state of spontaneous repetitive firing of action potentials corresponds to the dissipative structure with a stable limit cycle. The temporally coherent organization is realized through instability of the equilibrium point.     

Calcium content and net fluxes in squid giant axons

Axons freshly dissected from living specimens of the tropical squid Dorytheutis plei have a calcium content of 68 mumol/kg of axoplasm. Fibers stimulated at 100 impulses/s in 100 mM Ca seawater increase their Ca content by 150 mumol/kg.min; axons placed in 3 Ca (choline) seawater increase their Ca content by 12 mumol/kg.min. Axons loaded with 0.2--1.5 mmol Ca/kg of axoplasm extruded Ca with a half time of 15--30 min when allowed to recover in 3 Ca (Na) seawater. The half time for recovery of loaded axons poisoned with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and iodoacetic acid (IAA) is about the same as control axons. Axons placed in 40 mM Na choline seawater (to reduce chemical gradient for Na) or in 40 mM Na, 410 mM K seawater to reduce the electrochemical gradient for Na to near zero either fail to lose previously loaded Ca or gain further Ca.     

The periaxonal space of crayfish giant axons

The influence of the glial cell layer on effective external ion concentrations has been studied in crayfish giant axons. Excess K ions accumulate in the periaxonal space during outward K+ current flow, but at a rate far below that expected from the total ionic flux and the measured thickness of the space. At the conclusion of outward current flow, the external K+ concentration returns to normal in an exponential fashion, with a time constant of approximately 2 ms. This process is about 25 times faster than is the case in squid axons. K+ repolarization (tail) currents are generally biphasic at potentials below about -40 mV and pass through a maximum before approaching a final asymptotic level. The initial rapid phase may in part reflect depletion of excess K+. After block of inactivation and reversal of the Na+ concentration gradient, we could demonstrate accumulation and washout of excess Na ions in the periaxonal space. Characteristics of these processes appeared similar to those of K+. Crayfish glial cell ultrastructure has been examined both in thin sections and after freeze fracture. Layers of connective tissue and extracellular fluid alternate with thin layers of glial cytoplasm. A membranous tubular lattice, spanning the innermost glial layers, may provide a pathway allowing rapid diffusion of excess ions from the axon surface.