Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X-Ray Microprobe Analysis

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.     

Intracellular Concentrations of Major Ions in Rat Myelinated Axons and Glia: Calculations Based on Electron Probe X-Ray Microanalyses

Abstract: Electron probe x-ray microanalysis (EPMA) was used to measure water content (percent water) and dry weight elemental concentrations (in millimoles per kilogram) of Na, K, Cl, and Ca in axoplasm and mitochondria of rat optic and tibial nerve myelinated axons. Myelin and cytoplasm of glial cells were also analyzed. Each anatomical compartment exhibited characteristic water contents and distributions of dry weight elements, which were used to calculate respective ionized concentrations. Free axoplasmic [K⁺] ranged from ≈155 mM in large PNS and CNS axons to ≈120–130 mM in smaller fibers. Free [Na⁺] was ≈15–17 mM in larger fibers compared with 20–25 mM in smaller axons, whereas free [Cl^?] was found to be 30–55 mM in all axons. Because intracellular Ca is largely bound, ionized concentrations were not estimated. However, calculations of total (free plus bound) aqueous concentrations of this element showed that axoplasm of large CNS and PNS axons contained ≈0.7 mM Ca, whereas small fibers contained 0.1–0.2 mM. Calculated ionic equilibrium potentials were as follows (in mV): in large CNS and PNS axons, E_K = ?105, E_Na = 60, and E_Cl = ?28; in Schwann cells, E_K = ?107, E_Na = 33, and E_Cl = ?33; and in CNS glia, E_K = ?99, E_Na = 36, and E_Cl = ?44. Calculated resting membrane potentials were as follows (in mV, including the contribution of the Na⁺,K⁺-ATPase): large axons, about ?80; small axons, about ?72 to ?78; and CNS glia, ?91. E_Cl is more positive than resting membrane potential in PNS and CNS axons and glia, indicating active accumulation. Direct EPMA measurement of elemental concentrations and subsequent calculation of ionized fractions in axons and glia offer fundamental neurophysiological information that has been previously unattainable.     

Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na⁺,K⁺-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.     

Rubidium Uptake and Accumulation in Peripheral Myelinated Internodal Axons and Schwann Cells

Abstract: To study mechanisms of K⁺ transport in peripheral nerve, uptake of rubidium (Rb⁺), a K⁺ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K⁺ Ringer's solutions containing Rb⁺ (1–20 m M ) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb⁺ (Rb⁺_o) and exchanging it for internal K⁺. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1–10 m M Rb⁺_o concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb⁺ concentrations, which suggests involvement of a nonenzymatic process. At 20 m M Rb⁺_o, a differential stimulatory response was observed; i.e., axoplasmic Rb⁺ uptake velocities increased more than fivefold relative to the 10 m M rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb⁺_o uptake rate into axons and glia was completely inhibited by ouabain (2–4 m M ) exposure or incubation at 4°C. These results suggest that Rb⁺ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na⁺,K⁺-ATPase activity and implicate the presence of axonal- and glial-specific Na⁺ pump isozymes.     

Neuronal Na+, K+-ATPase in the peripheral nerve fibers

ATPase activity was localized by means of Wachstein-Meisel's method in rat sciatic nerve fibers. Using controls with ouabain, the presence of alpha + (neuronal) Na+, K+-ATPase was examined. The enzyme occurs in the ATPase reaction of the myelin-forming membranes, axoplasm and Schwann cell cytoplasm. Its presence in the Schwann cell plasma membrane is only admittable. The ATPase activity of the compact myelin and axolemma was exclusively of alpha + type of Na+, K+-ATPase.     

EFFECTS OF CALCIUM ION CONCENTRATION ON THE DEGENERATION OF AMPUTATED AXONS IN TISSUE CULTURE

Light and electron microscope studies were conducted on the nature of the degenerative changes in amputated nerve fibers of cultured rat sensory ganglia and on the effects of media with differing calcium concentrations upon these changes. With glucose-enriched Eagle's media (MEM) containing 1.6 mM calcium, the amputated myelinated and unmyelinated axons undergo a progressive granular disintegration of their axoplasm with collapse and fragmentation of myelin sheaths between 6 and 24 h after transection. With MEM containing only 25–50 µM calcium, the granular axoplasmic degeneration does not occur in transected fibers and they retain their longitudinal continuity and segmental myelin ensheathment for at least 48 h. Addition of 6 mM EGTA to MEM (reducing the estimated Ca⁺⁺ below 0.3 µM) results in the structural preservation of both microtubules and neurofilaments within transected axons. A transient focal swelling of amputated axons occurs, however, in cultures with normal and reduced calcium. These observations suggest that an alteration in the permeability of the axolemma is a crucial initiating event leading to axonal degenerative changes distal to nerve transection. The loss of microtubules and neurofilaments and the associated granular alterations of the axoplasm in transected fibers appears to result from the influx of calcium into the axoplasm.     

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.     

Element concentration changes in mitotically active and postmitotic enterocytes. An x-ray microanalysis study

Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.     

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.     

Effects of Increased Extracellular K on the Elemental Composition and Water Content of Neuron and Glial Cells in Leech CNS

Elemental (Na, Cl, K) and water contents of leech (Macrobdella decora) neurons and glial cells were determined under steady-state exposure to 4, 10, and 20 mM KCl concentrations (bathing media) using x-ray microanalysis for quantitative digital imaging of frozen hydrated and dried cryosections. Effects of furosemide, 5-hydroxytryptamine (5-HT), and ouabain on elemental distribution changes, induced by exposure to 20 mM K, were also determined. Results demonstrated that packet glial cells and neurons accumulated substantial amounts of K that appeared evenly distributed throughout the cytoplasm. Cell water content also increased as a function of increased cytoplasmic K so that the net effect was an unchanged wet-weight K concentration (expressed as millimoles per kilogram wet weight). Dry-weight Na and Cl concentration (expressed as millimoles per kilogram dry weight) increased slightly in glial cells; however, because cell water increased, both Na and Cl (wet-weight) concentrations decreased. Neurons, in contrast, had no significant change in either Na or K on a wet-weight basis, so a relatively constant Na/K ratio was maintained despite a small, but significant, increase in K (dry weight) and cell water. These increases, like those in packet glia, were a function of exposure to different concentrations of extracellular space K. These changes were completely abolished by 10(-4) M ouabain. Neither furosemide nor 5-HT appeared to affect neuronal or glial K wet-weight concentrations. These data show that both glial cells and neurons can act as substantial reservoirs for K while maintaining stable K concentrations (by altering cell water content and elemental composition). This process appears to depend on a functioning Na+, K+-ATPase system.     

Ultrastructural distribution of Ca++ within neurons. An oxalate pyroantimonate study

We used the oxalate-pyroantimonate technique to determine the ultrastructural distribution of Ca++ in neurons of the rat sciatic nerve. The content of the precipitate was confirmed by X-ray microanalysis and appropriate controls. In the cell bodies of the dorsal root ganglia, Ca++ precipitate was found in the Golgi, mitochondria, multivesicular bodies and large vesicles of the cytoplasm but not in lysosomes, and was prominently absent from regions of rough endoplasmic reticulum and ribosomes. It was seen in the nucleus but not in the nuclear bodies or nucleolus. Within the axon itself, Ca++ precipitate was also found sequestered in mitochondria and smooth endoplasmic reticulum. In addition Ca++ precipitate found diffusely throughout the axoplasm exhibited a discrete and heterogeneous distribution. In myelinated fibers the amount of precipitate decreased predictably in the axoplasm beneath the Schmidt-Lanterman cleft and in the paranodal regions at the nodes of Ranvier. This correlated with the presence of dense precipitate in the Schmidt-Lanterman cleft themselves and in the paranodal loops of myelin. Intracytoplasmic ionic Ca++ is maintained at 10(-7) M by balanced processes of influx, sequestration and extrusion. The irregular distribution of Ca++ precipitate in the axoplasm of myelinated fibers suggests that there may be specific regions of preferential efflux across the axolemma.     

Ultrastructural changes of human sural nerves in the neuropathy induced by intrauterine methylmercury poisoning (so-called fetal Minamata disease).

Biopsy of the sural nerve was performed on three patients with severe Minamata disease of more than 10 years duration. There were so many unmyelinated and poorly myelinated nerve fibers that myelinated fibers scattered irregularly in small numbers or in groups of peculiar features in the intraneural bundle. Abnormaly thin or poorly formed myelin sheaths were noticed. Incomplete myelination and abnormal myelination varied in size and shape appeared as fetal anomaly. Regenerated axons extremely small in size remained singly or in groups following regenerative sprouting. Sometimes, extremely small axons with normal myelination were noticeable, while the axons were lost, leaving myelin sheaths. Axons occasionally contained increased neurofilaments. Schwann cells were not so increased as in adult Minamata disease. Degenerative changes of nerve fibers still proceeded, presumably because the patients lived in the mercury-contaminated district. Myelin degenerations and glycogen deposits in the axoplasm were identified.     

Influence of soil oxygen and soil water on the accumulation of nutrients in avocado seedlings (Persea Americana mill.)

Summary This experiment was conducted in a greenhouse to study the influence of 2 soil-oxygen levels and 4 irrigation levels on the plant response, root decay, concentrations of 12 nutrients, as well as on total amounts of nutrients per avocado seedling (Persea americana Mill.).Reduced soil-oxygen supply to the roots significantly reduced the amount of dry weight per seedling, increased percentage of root decay, and reduced the concentrations of N, P, K, Ca, Mg, and B in the tops, while Na and Fe were increased. Concentrations of K, Mg, Na, and Cl in the roots were decreased, while N and Ca were increased with decreased soil oxygen supply to the roots. Total amounts of N, P, Ca, Mg, Na, and Cl per seedling were decreased with the low soil-oxygen supply to the roots.Only slight differences in dry weight of the tops of seedlings were found. The highest degree of root decay was caused by the irrigation treatment where a water table was present. In the tops, concentrations of N, P, K, Mg, Na, Zn, Cu, Mn, B, and Fe were significantly influenced by differential irrigation treatments; in the roots, concentrations of P, K, Ca, Mg, Na, and Cl were also significantly influenced; and total amounts of N, P, Mg, and Cl the whole seedling were likewise significantly influenced.Significant interactions were noted between the soil-oxygen and irrigation treatments on the dry weight of tops, roots, and total amounts of dry weight produced per seedling. The lowest amount of dry weight of roots and the highest degree of root decay were found in the avocado seedlings grown under low soil-oxygen supply and the irrigation treatment where a water table was present. Several significant interactions between soil oxygen and irrigation on the concentrations of N, P, K, Ca, Zn, and Mn are discussed.University of California, Citrus Research Center and Agricultural Experiment Station, Riverside, California. The research reported in this paper was supported in part by NSF Grant GB-5753x.     

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.     

2,5-Hexanedione Alters Elemental Composition and Water Content of Rat Peripheral Nerve Myelinated Axons

Abstract: Effects of 2,5-hexanedione on elemental concentrations and water content of peripheral nerve myelinated axons were determined using electron probe x-ray microanalysis. Axons (small, medium, and large) were analyzed in unfixed cryosections from rat tibial and proximal sciatic nerve samples. Animals were intoxicated with 2,5-hexanedione by two dosing paradigms: intraperitoneal or oral. Regardless of the route of exposure, internodal axoplasm of small and medium axons from both nerve regions exhibited selective, progressive reductions in dry weight K concentrations and water content. When calculated on a wet weight basis, K levels were comparable to or slightly above control values in tibial nerve, whereas in sciatic nerve, small transient decreases in wet weight K were evident. These changes in K and water correlated with the development of axonal atrophy. The wet and dry weight internodal elemental changes reported here do not suggest a metabolic or axolemmal defect, but rather imply a homeostatic response possibly related to the process of axonal atrophy. Giant axonal swellings were primarily associated with oral 2,5-hexane-dione intoxication, and corresponding analyses revealed few changes in element or water content compared with control. The absence of significant alterations in these swellings is consistent with mechanical expansion of the axon probably as a function of accumulating neurofilaments.     

X-ray microprobe analysis of epithelial calcium transport

The sternal epithelium of Porcellio scaber was used as a novel model to study the subcellular elemental distribution in control and Ca(2+)-transporting stages in situ. The anterior sternal epithelium (ASE) is specialized for transport of cuticular Ca to sternal CaCO(3) deposits during premolt, and from these deposits during intramolt. The less specialized posterior sternal epithelium transports Ca(2+) to and from the cuticle. In the ASE cells basal [Na], [Cl], and [Mg] are higher than in the apical side. The basal [Na] increases from 105 to 173 mmol/kg dry mass between control and Ca(2+)-transporting stages, accompanied by a decrease in [Cl] and [K]. The [Mg] increases, suggesting transepithelial Mg(2+)-transport. Cytosolic [Ca] varied insignificantly between 4.5 and 5.7 mmol/kg dry mass, however, the number of Ca hot-spots with concentrations between 15 and 50 mmol/kg dry mass increased during transport. Mitochondrial [Ca] decreased in the ASE from 3.3 in the control to 1.0 in the late premolt and to 2.0 mmol/kg dry mass in the intramolt stage. The results suggest Na(+)-dependent mechanisms for transcellular Ca(2+)-transport and the presence of Ca(2+)-binding proteins. Organelles, probably the smooth endoplasmic reticulum, sequester Ca(2+) during intracellular Ca(2+)-transport. A role of mitochondria as a storage site for cuticular Ca is excluded.     

ELEMENTAL COMPOSITION,CORRELATIONS, AND RATIOS WITHIN A POPULATION OF STAURASTRUM PLANCTONICUM (ZYGNEMATALES): AN X-RAY MICROANALYTICAL STUDY1

Individual cells of Staurastrum planctonicum (Teil.) within a mixed freshwater phytoplankton sample were analyzed by scanning electron microscope X-ray microanalysis to determine their elemental composition. X-ray emission spectra routinely showed clear peaks of P, S, and Cl, plus monovalent (Na, K, and divalent (Mg, Ca) cations. Si and Cu were also present in lower quantities. Concentrations of individual elements (expressed as mmol.kg⁻¹ dry weight) varied widely among cells, with values over the sample population approximating to a normal distribution. Although intracellular anion and cation levels varied considerably, significant correlations occurred between concentrations of monovalent and divalent cations (mean ratio 1.4), major diffusible anions and cations (mean ratio 1,2), and total levels of electropositive and electronegative elements (mean ratio 1.2). The monovalent cations of K and Na occurred at a mean ratio of 1.2 and were not significantly correlated. Concentrations of individual elements (except Si) showed clear positive correlations within the analyses, with 12 highly significant (99% probability) correlations out of 36 possible combinations. Principal factor analysis showed that elemental correlations were determined by two major factors, with two resulting groups of elements—(Na, S, Cl, Ca, Cu) and (Mg, P, K). Statistical relationships between elements followed a clear correlation pattern, which retained its characteristics even when elemental concentrations were expressed per unit P rather than per unit dry weight. Elemental concentrations were closely similar in matching, but not nonmatching, smicells. The statistical pattern of elemental associations noted in Staurastrum parallels that seen in X-ray micro-analytical studies of other algae but differs in detail. This pattern of statistical associations has biological implications in terms of cell compartmentation, characterization of different cell types, and cell interactions with their environment.     

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.     

Localization of Phospholipid Synthesis to Schwann Cells and Axons

Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glycerol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent-shaped perinuclear cytoplasm of myelinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt-Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholipids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: myelinating Schwann cells dominate phospholipid and protein synthesis, myelinated axons selectively incorporate tritiated inositol, phospholipid precursors label myelin sheaths and myelinated axons better than proline.     

Stimulus-permeability coupling in rat pulmonary macrophages challenged by Pseudomonas aeruginosa

Summary Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 501 for 1 h at 37° C. A 1-mm³ pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-m cryosections were cut at -100° C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus and cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p<0.001) whereas Cl was double (p<0.001), K was 29% lower (p<0.001), and Ca was 4 times higher (p<0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrate elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.