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SUMMARY1. Until recently it was believed that axoplasmic transport in vitro was not affected by Ca2+, transport being normal in Ca2+-free medium. This was found due to the presence of the relatively impermeable perineurial sheath around the nerve trunks. Using a desheathed cat peroneal nerve preparation, axoplasmic transport was shown to require an adequate level of Ca2+ in the external medium. In a buffered Ca2+-free medium, transport began to decline within 30 min and a complete block occurred in 2-6 hr. A concentration of 5 mM-Ca2+ added to a buffered isotonic sucrose or NaCl solution was able to maintain transport. With lower concentrations of Ca2+ of 1 5-3'0 mm, those usually present in the extracellular fluid or in a Ringer medium, some impairment of transport was seen but the addition of 4 mM-K+ restored the normal pattern of axoplasmic transport. With Ca2+ concentrations below 0 75 mm, however, 4 mM-K+ was unable to sustain transport.2. Potassium by itself at a concentration of 4 mm when added to a buffered isotonic sucrose or NaCl medium was unable to prolong the time of transport block beyond that seen in buffered isotonic NaCl or sucrose solutions. In concentrations of K+ up to 25 mm, 1 5-5 mM-Ca2+ was required for normal transport. With moderately higher concentrations of K+ in the range of 50-100 mm, normal appearing transport was seen with or without Ca2+. This was seen whether or not Na+ was present in the medium. At higher levels of K+, 120-150 mm, decreased transport was seen, with or without the addition of either 15 mM-Na+ or Ca2+ in concentrations of 1 5-3-0 mm.3. While Mg2+ could not substitute completely for Ca2+ in maintaining transport, it was able to prolong the time before block occurred. An extra 30-60 min of downflow was seen when 5 mMMg2+ was added to a buffered isotonic NaCl medium. Magnesium also acts synergistically with Ca2+. Concentration of Ca2+ as low as 0-25 mm was, with the addition of 1-5 mM-Mg2+, able to maintain transport.4. The results are interpreted in the light of studies of the mechanism of Ca2+ regulation known to occur in giant nerve fibres and other cells controlling the level of free Ca2+. The relationship of Ca2+ to the mechanism considered to underlie axoplasmic transport in nerve fibres is also discussed.

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1-100 microM was examined. The relative order of potency in blocking axoplasmic transport was VCR greater than VLB greater than VDS at a concentration of 25 microM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block to the transport mechanism is discussed.

Using the desheathed nerve preparation, a pyroantimonate precipitation method was used to examine the distribution of electron-dense particles seen in various organelles of the nerve fibers following exposure of nerve to various levels of Ca2+ in vitro. The presence of Ca2+ in the electron-dense particles was indicated by their extraction with EGTA and by the use of energy-dispersive X-ray microanalysis. In normal Ringer or in a Ca2+ -free medium, electron-dense particles were seen associated with the outer membrane of the mitochondria, with the smooth endoplasmic reticulum (SER), along the axolemma and yet others scattered throughout the axoplasm. When nerves were incubated in media containing higher than normal concentrations of 20-60 mM Ca2+, an increase in the number of such electron-dense particles was seen in the axoplasm and within the mitochondrial matrix. Nerves loaded with a high concentration of 60mM Ca2+ could be depleted of these particles after transfer to a Ca2+ -free or low Ca2+ Ringer medium. The sequestration of Ca2+ in axonal organelles is discussed with respect to Ca2+-regulatory mechanisms in the axon needed to maintain a low level of Ca2+ which is optimal for the support of axoplasmic transport.

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