Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

Trophoblast specialisations during pregnancy in the tammar wallaby, Macropus eugenii: a morphological and lectin histochemical study.

INTRODUCTION: The tammar wallaby has a short gestation (26.5 days) and vascular modifications to expedite transport during that brief pregnancy. Here we examine trophoblast structural attributes that would facilitate materno-fetal exchange. MATERIALS AND METHODS: Four specimens of Macropus eugenii between days 23 and 26 gestation were examined using electron microscopy and 24 lectins to characterise glycosylated secretions and their internalisation. RESULTS: Two trophoblast phenotypes were found, flattened cells generally in contact with the underlying uterine epithelium and giant cells associated with histiotrophe. The latter appeared to penetrate uterine clefts, occasionally detach and become necrotic. Lectin histochemistry and ultrastructure indicated the presence of many lysosomes and residual bodies especially in trophoblast giant cells; these contained glycans, mainly apically, which were also detected in secretions and cell debris. Trophoblast basal membranes bore extensive filopodia. Giant cells were less common in vascular trilaminar areas and here the trophoblast barrier became thinner near term. DISCUSSION: Loss of Maackia amurensis agglutinin binding suggested cleavage of terminal sialic acid residues as an early post-internalisation event in the trophoblast. Lectin staining indicated degradation occurred in an apical-basal direction, and the heavily glycosylated basal membrane appeared specialised for transport out of the cell. CONCLUSION: Granules seen ultrastructurally and histochemically, particularly in giant trophoblast cells of the bilaminar area, suggest that internalised histiotrophe is broken down here and nutrients transferred to the embryo via the specialised basal plasma membrane. The trilaminar vascular area contained mostly flattened trophoblast cells, supporting the suggestion that gaseous exchange is its primary function.

Memantine block depends on agonist presentation at the NMDA receptor in substantia nigra pars compacta dopamine neurones.

NMDA glutamate receptors (NMDARs) have critical functional roles in the nervous system but NMDAR over-activity can contribute to neuronal damage. The open channel NMDAR blocker, memantine is used to treat certain neurodegenerative diseases, including Parkinson's disease (PD) and is well tolerated clinically. We have investigated memantine block of NMDARs in substantia nigra pars compacta (SNc) dopamine neurones, which show severe pathology in PD. Memantine (10 µM) caused robust inhibition of whole-cell (synaptic and extrasynaptic) NMDARs activated by NMDA at a high concentration or a long duration, low concentration. Less memantine block of NMDAR-EPSCs was seen in response to low frequency synaptic stimulation, while responses to high frequency synaptic stimulation were robustly inhibited by memantine; thus memantine inhibition of NMDAR-EPSCs showed frequency-dependence. By contrast, MK-801 (10 µM) inhibition of NMDAR-EPSCs was not significantly different at low versus high frequencies of synaptic stimulation. Using immunohistochemistry, confocal imaging and stereological analysis, NMDA was found to reduce the density of cells expressing tyrosine hydroxylase, a marker of viable dopamine neurones; memantine prevented the NMDA-evoked decrease. In conclusion, memantine blocked NMDAR populations in different subcellular locations in SNc dopamine neurones but the degree of block depended on the intensity of agonist presentation at the NMDAR. This profile may contribute to the beneficial effects of memantine in PD, as glutamatergic activity is reported to increase, and memantine could preferentially reduce over-activity while leaving some physiological signalling intact.

In vitro osteoblast response to ferritic stainless steel fiber networks for magneto-active layers on implants.

The use of a porous coating on prosthetic components to encourage bone ingrowth is an important way of improving uncemented implant fixation. Enhanced fixation may be achieved by the use of porous magneto-active layers on the surface of prosthetic implants, which would deform elastically on application of a magnetic field, generating internal stresses within the in-growing bone. This approach requires a ferromagnetic material able to support osteoblast attachment, proliferation, differentiation, and mineralization. In this study, the human osteoblast responses to ferromagnetic 444 stainless steel networks were considered alongside those to nonmagnetic 316L (medical grade) stainless steel networks. While both networks had similar porosities, 444 networks were made from coarser fibers, resulting in larger inter-fiber spaces. The networks were analyzed for cell morphology, distribution, proliferation, and differentiation, extracellular matrix production and the formation of mineralized nodules. Cell culture was performed in both the presence of osteogenic supplements, to encourage cell differentiation, and in their absence. It was found that fiber size affected osteoblast morphology, cytoskeleton organization and proliferation at the early stages of culture. The larger inter-fiber spaces in the 444 networks resulted in better spatial distribution of the extracellular matrix. The addition of osteogenic supplements enhanced cell differentiation and reduced cell proliferation thereby preventing the differences in proliferation observed in the absence of osteogenic supplements. The results demonstrated that 444 networks elicited favorable responses from human osteoblasts, and thus show potential for use as magnetically active porous coatings for advanced bone implant applications.

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function.

The binding of Bcl-2 to Beclin-1 reduces Beclin-1's capacity to induce autophagy. Here, we have tested whether the interaction is reciprocated by loss of Bcl-2's anti-apoptotic function. We targeted Bcl-2 to mitochondria or endoplasmic reticulum (ER) and induced apoptosis using several apoptotic stimuli that initiate ER and/or mitochondrial signaling pathways (UV radiation, TNF and cycloheximide, staurosporine, thapsigargin and tunicamycin). When Beclin-1 and Bcl-2 were expressed together in HeLa cells, Beclin-1 (but not Beclin-1 lacking the Bcl-2-binding domain) followed Bcl-2 to the appropriate organelle with complete or near-complete overlap (comprising 60 and 30% of cells, respectively). The interaction between Beclin-1 and Bcl-2 was verified by immunoprecipitation, and a membrane-proximate localization of Beclin-1 was shown by immunoelectron microscopy. Apoptosis was followed by measuring changes in nuclear morphology, caspase-3 activity, poly-ADP-ribose polymerase cleavage or punctation of mRFP-Bax on mitochondria. Binding of Beclin-1 to Bcl-2 did not modify apoptosis irrespective of Bcl-2 concentration, location or apoptotic stimulus. A similar result was obtained in Atg5-/- cells that are autophagy-deficient, arguing against compensation for the loss of protection by Bcl-2 by autophagy-mediated survival induced by Beclin-1. Hence, although Beclin-1 contains a BH3-only motif typical of pro-apoptotic proteins, it is a negligible modulator of Bcl-2's anti-apoptotic function.

Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability.

Synthetic colloid and gel hydroxyapatite (HA) nanoparticles (NPs) were spray dried to form microparticles (MPs). These are intended for use as slow release vaccine vectors. The physico-chemical properties of gel and colloid NPs and MPs were compared to those of HA obtained commercially. Their cytotoxicity to human monocytes'-derived macrophages (HMMs) was assessed in vitro using a range of techniques. These included the MTT assay, LDH leakage and a confocal based live-dead cell assay. Cytotoxicity differed significantly between preparations, with the suspended gel preparation being the most toxic (31-500 microg/ml). Other preparations were also toxic but only at higher concentrations (>250 microg/ml). Transmission electron microscopy (TEM) and stereology showed variable cellular uptake and subsequent dissolution of the various forms of HA. We have demonstrated that HA particle toxicity varied considerably and that it was related to their physico-chemical properties. Cell death correlated strongly with particle load. The intracellular dissolution of particles as a function of time in HMM suggests that increased cytoplasmic calcium load is likely to be the cause of cell death. Some HA NPs eluded the phagocytic pathway and a few were even seen to enter the nuclei through nuclear pores.

Application of environmental scanning electron microscopy to determine biological surface structure.

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification.

BACKGROUND: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five gamma-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca(2+), calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. METHODS AND RESULTS: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca(2+) concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K(1) showed decreased calcification as compared to controls. With conformation-specific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the gamma-carboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. CONCLUSIONS: These data demonstrate that both gamma-glutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation.

BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.

Clozapine and haloperidol differentially regulate dendritic spine formation and synaptogenesis in rat hippocampal neurons.

Antipsychotic drugs are the primary therapeutic treatment for schizophrenia. In addition to their dopaminergic/serotonergic function, atypical antipsychotics differ from conventional antipsychotics in the way they affect glutamatergic receptor function. A cellular correlate of this may be the modulation of dendritic spines (DS). Here, we demonstrate that in rat dissociated hippocampal neurons 1.0 microM clozapine administration increased DS-enriched protein spinophilin by 70%, increased post-synaptic protein shank1a puncta density by 26% and increased overall primary dendrite DS density by 59%. Filopodia and mushroom DS were particularly affected by clozapine. Conversely, 0.1 microM haloperidol decreased spinophilin protein by 40%, caused a 25% decrease in shank1a puncta and reduced the numbers of filopodia. In contrast, neither haloperidol nor clozapine induced any change in the levels of the pre-synaptic protein synapsin. This indicates that clozapine and haloperidol differentially regulate DS and post-synaptic plasticity. These findings may provide a molecular and cellular correlate to the superior therapeutic profile of clozapine when compared with haloperidol.

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells.

Decreases in the intracellular concentrations of both K(+) and Cl(-) have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied.

Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics.

The incorporation of silicate into hydroxyapatite (HA) has been shown to significantly increase the rate of bone apposition to HA bioceramic implants. However, uncertainty remains about the mechanism by which silicate increases the in vivo bioactivity of HA. In this study, high-resolution transmission electron microscopy was used to observe dissolution from HA, 0.8 wt% Si-HA and 1.5 wt% Si-HA implants after 6 and 12 weeks in vivo. Our observations confirmed that defects, in particular those involving grain boundaries, were the starting point of dissolution in vivo. Dissolution was observed to follow the order 1.5 wt% Si-HA>0.8 wt% Si-HA>pure HA and it was found to be particularly prevalent at grain boundaries and triple-junctions. These observations may help to explain the mechanism by which silicate ions increase the in vivo bioactivity of pure HA, and highlight the enhanced potential of these ceramics for biomedical applications.

A reappraisal of the contrasting morphological appearances of villous cytotrophoblast cells during early human pregnancy; evidence for both apoptosis and primary necrosis.

Villous cytotrophoblast cells display a range of morphological appearances that are assumed to reflect different stages of differentiation. Here we demonstrate that apoptosis and primary necrosis can also occur in these cells during normal early pregnancy, and should be included in the list of possible phenotypes. Samples from 30 placentae of 6-15 weeks gestational age were examined. Cytotrophoblast cells displaying highly condensed chromatin, but no karyorhexis, were observed detached from the basement membrane, and represented 0.49% (s.d.+/-0.36) of the total population. Their cytoplasm was heavily vacuolated, and their mitochondria swollen, indicating secondary necrosis. By contrast, extremely pale-staining cells with large rounded nuclei (volume-weighted mean volume 471.6 microm(3) compared to 250.1 microm(3) for euchromatic cells) were frequently observed (5.97% of total, s.d.+/-4.31). These cells displayed loss of euchromatin, a paucity of cytoplasmic organelles, and swelling of the mitochondrial intracristal space and endoplasmic reticulum. Nuclei of these cells displayed a significantly higher level of gold labelling using the TUNEL technique compared to euchromatic nuclei [1.0 particles/microm(2) (s.d.+/-0.13) vs 0.12 particles/microm(2) (s.d.+/-0.03),P< 0.05], confirming increased DNA fragmentation. We conclude that these cells are undergoing primary necrosis. The stimulus for both forms of cell death remains unknown, but may be associated with syncytiotrophoblastic stress.

Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis.

BACKGROUND: Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. METHODS AND RESULTS: In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase-polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased approximately 3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. CONCLUSIONS: These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.

Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.

Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with either of two cell surface receptors, TNFR-1 and TNFR-2. The levels and regulation of expression of these two receptors has been extensively analyzed in cultured cells, but little is known of TNFR expression in situ. We analyzed the expression of TNFR-1 and -2 in normal human renal kidney and in renal transplants undergoing acute cellular rejection. Immunohistochemistry and immunogold electron microscopy indicated a strong expression of TNFR-1 on the endothelium of glomeruli of normal kidney. Immunogold colocalization for TNFR-1 and a marker of the trans-Golgi network (TGN-46) demonstrated TNFR-1 within the Golgi complex in endothelial cells in normal kidney, confirming our previous studies with cultured cells. TNFR-1 expression was lost in glomeruli from acutely rejecting kidney, but TNFR-1 was detected in abundance on infiltrating leukocytes in the interstitium of allografts with acute rejection. In contrast, TNFR-2 was demonstrated predominantly in epithelial cells of distal convoluted tubule (DCT) in acute rejection kidney near TNF-expressing leukocytes. TNF was absent in normal kidney, but present in rejecting allograft. TNF was found in infiltrating leukocytes and in adjacent tubular epithelial cells. In situ hybridization showed TNFR-1 mRNA within the endothelium of the glomeruli and of a few arterioles in normal kidney, whereas TNFR-2 mRNA was seen in tubular epithelial cells of the DCT in acute transplant rejection. These data reveal that there is both differential expression and regulation of the two TNF receptors in human kidney.

Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

Calcium waves induced by hypertonic solutions in intact frog skeletal muscle fibres.

1. Regenerative Ca2+ waves and oscillations indicative of calcium-induced calcium release (CICR) activity were induced in fully polarized, fluo-3-loaded, intact frog skeletal muscle fibres by exposure to hypertonic Ringer solutions. 2. The calcium waves persisted in fibres exposed to EGTA-containing solutions, during sustained depolarization of the membrane potential or following treatment with the dihydropyridine receptor (DHPR)-blocker nifedipine. 3. The waves were blocked by the ryanodine receptor (RyR)-specific agents ryanodine and tetracaine, and potentiated by caffeine. 4. In addition to these pharmacological properties, the amplitudes, frequency and velocity of such hypertonicity-induced waves closely resembled those of Ca2+ waves previously described in dyspedic skeletal myocytes expressing the cardiac RyR-2. 5. Quantitative transmission and freeze-fracture electronmicroscopy demonstrated a reversible cell shrinkage, transverse (T)-tubular luminal swelling and decreased T-sarcoplasmic reticular (SR) junctional gaps in fibres maintained in and then fixed using hypertonic solutions. 6. The findings are consistent with a hypothesis in which RyR-Ca2+ release channels can be partially liberated from their normal control by T-tubular DHPR-voltage sensors in hypertonic solutions, thereby permitting CICR to operate even in such fully polarized skeletal muscle fibres.

In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies.

Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2',7'-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and alpha-phenyl-N-tert-butylnitrone reduced levels of oxidative stress after reoxygenation. These changes are consistent with an ischemia-reperfusion injury, and mirror those seen in preeclampsia. Consequently, in vitro hypoxia/reoxygenation may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy.

Normal conduction of surface action potentials in detubulated amphibian skeletal muscle fibres.

1. The influence of the transverse (T) tubules on surface action potential conduction was investigated by comparing electrophysiological and confocal microscopic assessments of tubular changes in osmotically shocked and control fibres from frog sartorius muscle. 2. The membrane-impermeant fluorescent dye, di-8-ANEPPs spread readily from the bathing extracellular solution into the tubular membranes in control, intact fibres. Prior exposure of muscles to a hypertonic glycerol-Ringer solution, its replacement by an isotonic Ca(2+)-Mg(2+) Ringer solution and cooling sharply reduced such access. In contrast, dye application in the course of this osmotic shock procedure stained the large tubular vacuoles hitherto associated with successful muscle detubulation. 3. Conduction velocities in intact, control fibres (1.91 +/- 0.048 m s(-1), mean +/- S.E.M., n = 32 fibres) agreed with earlier values reported at room temperature (18-21 degrees C) and were unaffected by prior episodes of steady cooling to 8-10 degrees C (1.91 +/- 0.043 m s(-1), n = 30). 4. Cooling to 11.5 degrees C reduced these velocities (1.47 +/- 0.081 m s(-1), n = 25) but action potential waveforms still included early overshoots and the delayed after-depolarizations associated with tubular electrical activity. 5. In contrast, action potentials from cooled, superficial fibres in osmotically shocked muscles lacked after-depolarization phases implying tubular detachment. Their mean conduction velocities (1.62 +/- 0.169 m s(-1), n = 25) were not significantly altered from values obtained in untreated controls or in intact fibres in muscle similarly treated with glycerol, in direct contrast to earlier results. 6. Cooling produced similar reductions in maximum rates of voltage change dV/dt in action potentials from all fibre groups with lower rates of change shown by detubulated fibres. 7. Use of an antibody to a conserved epitope of the alpha-subunit of voltage-gated sodium channels suggested a concentration of sodium channels close to the mouths of the T tubules. 8. These electrophysiological and anatomical findings are consistent with a partial independence of electrical events in the transverse tubules from those responsible for the rapid conduction of surface regenerative activity. 9. The findings are discussed in terms of a partial separation of the electrical activity propagated over the surface membrane, from the initiation of propagated activity within the T tubules, by the triggering of the sodium channels clustered selectively around the mouths of the T tubules.