Mechanisms of Injury-Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium-Calcium Exchange

Abstract: To investigate the route of axonal Ca²⁺ entry during anoxia, electron probe x-ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na⁺ and Ca²⁺ movements. Perfusion of nerve segments with zero-Na⁺/Li⁺-substituted medium and Na⁺ channel blockade by tetrodotoxin (1 µM) prevented anoxia-induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero-Ca²⁺/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na⁺-Ca²⁺ exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L-type Ca²⁺ channel blocker, did not alter anoxia-induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero-Ca²⁺ and zero-Na⁺ solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na⁺ enters axons via voltage-gated Na⁺ channels and that subsequent increases in axoplasmic Na⁺ are coupled functionally to extraaxonal Ca²⁺ import. Intracellular Na⁺-dependent, extraaxonal Ca²⁺ entry is consistent with reverse operation of the axolemmal Na⁺-Ca²⁺ exchanger, and we suggest that this mode of Ca²⁺ influx plays a general role in peripheral nerve axon injury.     

Antibodies That Distinguish between the Serine-158 Phospho- and Dephospho-Form of Spinach Leaf Sucrose-Phosphate Synthase

Serum antibodies were raised against a synthetic peptide corresponding to the amino acid sequence surrounding the major inactivating phosphorylation site (serine-158) of spinach (Spinacia oleracea) leaf sucrose-phosphate synthase (SPS). The anti-peptide antibodies precipitated highly activated SPS preferentially to ATP-inactivated SPS and interacted only weakly with the sodium dodecyl sulfate-denatured enzyme bound to a membrane. The antibodies blocked phosphorylation but not dephosphorylation of SPS. Highly activated SPS was not entirely dephosphorylated and ATP-inactivated SPS was not completely phosphorylated on serine-158, as indicated by the sensitivities of immunopurified serine-158 phospho- and dephospho-SPS to inhibition by inorganic phosphate. The anti-peptide antibodies can be used to detect changes in the phosphorylation state of serine-158, and they are useful to purify and characterize distinct kinetic forms of SPS.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase)

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the sialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens$\text{N-}\text{acetylneuraminate}$ glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspension medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca²⁺ in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that a restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

Persistent atrial paralysis: Case report with light microscopy and ultrastructural analyses

Persistent atrial paralysis in a patient with complete heart block and mild mitral insufficiency is presented. Left atrial specimens obtained during implantation of a permanent cardiac pulse generator showed evidence of hypertrophy and fibrosis; subcellular degenerative changes ranged from near normal to irreversible, thus suggesting that atrial paralysis may be due to the replacement of normal atrial muscle with nonfunctional fibrous tissue.     

Clonazepam and dopamine-related stereotyped behavior

The effect of clonazepam on behavioral stereotypy induced by a number of dopaminergic agonists was investigated. Clonazepam inhibited levodopa- and amphetamine-induced behavior in guinea pigs but did not alter lergotrile- or apomorphine-induced stereotypy. The results suggest that clonazepam influences the central dopaminergic system through a direct effect of dopaminergic presynaptic mechanisms.