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.

Loop diuretics inhibit detubulation and vacuolation in amphibian muscle fibres exposed to osmotic shock.

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detubulation following osmotic shock, was investigated in amphibian skeletal muscles. These were exposed to a glycerol-Ringer solution (18 min), an isotonic Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 microM) to these solutions sharply reduced the incidence of detubulation, assessed by abolition or otherwise of action potential after-depolarisations, from 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 microM bumetanide). It dramatically reduced the number and fraction of muscle volume occupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horseradish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, similarly was reduced with 2 microM bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulation protocol. Thus, it exerted its maximum effect when added to the glycerol-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas fibre diameters measured using scanning electron microscopy returned to normal during glycerol treatment relative to those of control fibres left in isotonic Ringer, addition of 2.0 microM bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics. furosemide and ethacrynic acid similarly influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introduction of extracellular glycerol. This might then enable the subsequent Na-K-ATPase dependent water extrusion that produces the tubular distension (vacuolation) and detachment (detubulation) following glycerol withdrawal, phenomena also observed in muscular dystrophy.

Endocytosis and vesicular trafficking of immune complexes and activation of phospholipase D by the human high-affinity IgG receptor requires distinct phosphoinositide 3-kinase activities.

FcgammaRI, the human high-affinity IgG receptor, is responsible for the internalization of immune complexes and their subsequent targetting to the lysosomes for degradation. We show here that aggregation of FcgammaRI by surface immune complexes in interferon-gamma-primed U937 cells causes the transient appearance of swollen vacuolar structures, probably swollen late endosomes, which disappear as the immune complexes are degraded. Wortmannin and LY294002, specific inhibitors of phosphoinositide 3-kinases (PI 3-kinases), delay the disappearance of these structures and also correspondingly inhibit degradation of FcgammaRI-mediated immune complexes. In addition these inhibitors delay the initial phase of FcgammaRI-mediated endocytosis of immune complexes and block the activity of FcgammaRI-stimulated phospholipase D, an enzyme that has previously been implicated in membrane-trafficking events. p85 is the regulatory subunit of PI 3-kinase. A p85-dependent PI 3-kinase was shown to be involved in the initial phase of FcgammaRI-mediated endocytosis, but not in the trafficking of immune complexes for degradation or the activation of phospholipase D. The results presented here show a role for a p85-independent PI 3-kinase in regulating the trafficking of FcgammaRI-mediated immune complexes, either directly or as a result of the activation of phospholipase D, and a distinct role for a p85-dependent PI 3-kinase isoform in the initial phases of FcgammaRI-mediated internalization of immune complexes.

The effects of transport perturbations on the homeostasis of erythrocytes.

The control of erythrocyte volume, pH, membrane potential and ion content results from the interaction of many passive and active transport systems, cytoplasmic buffers, and from the charge and osmotic properties of haemoglobin and other impermeant solutes. The complexity of the system is such that the understanding of cell responses to experimental, physiological and pathophysiological challenges is beyond intuitive grasp. Mathematical models of erythrocyte and reticulocyte homeostasis have delivered a wealth of novel and unexpected predictions that have been confirmed experimentally. Those concerning effects of Ca(2)+ and K+ permeabilization on cell volume, pH and osmolality have helped solve long-standing issues on the pathophysiology of sickle-cell dehydration and will be briefly reviewed here. To study the effects of parasite growth and of new permeation pathways (NPP) on host cell homeostasis, we have developed a model of a Plasmodium falciparum- infected erythrocyte. Modelling NPP to fit reported changes in both Na+/K+ fluxes and gradients predicted large variations in host cell haemoglobin concentration, [Hb]. However, preliminary estimates seem to indicate that host cell [Hb] is conserved throughout the parasite's asexual cycle, suggesting that the properties of the NPP vary in subtle, stage-dependent ways.

The tubular vacuolation process in amphibian skeletal muscle.

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes 'vacuolation' in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface ('detubulation'), particular if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for he first time two types of vacuole, 'open' and 'closed', whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed closed to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. 'Open' vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. 'Closed' vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca/+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by 'open' vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole 'closure' took place well after formation of 'open' vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of 'open' vacuole formation. These findings identify electrophysiological 'detubulation' of striated muscle with 'closure' of initially 'open' vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.

Binding of monomeric immunoglobulin G triggers Fc gamma RI-mediated endocytosis.

Cross-linking of leukocyte Fc receptors specific for IgG (Fc gamma Rs) by multivalent IgG complexes triggers a wide range of immune functions. Many of these responses can also be stimulated in vitro using anti-Fc gamma R monoclonal antibody-containing complexes. This observation has suggested that cross-linking is the key event and that binding of IgG, which in itself does not elicit a response, is functionally passive. However, in this study we show that binding of monomeric IgG to the human high affinity receptor, Fc gamma RI, is itself sufficient to permit the receptor to enter an internalization-recycling pathway, which has a small intracellular pool. Unoccupied Fc gamma RI is not internalized and recycled in this manner. This finding may be explained by the previous observation that there is a physical association between Fc gamma RI and the cytoskeletal component, actin-binding protein (non-muscle filamin; ABP-280), which is disrupted upon IgG binding. Thus, in the absence of IgG, Fc gamma RI may be physically excluded from the endocytic pathway by tethering to the cytoskeleton. The role of cross-linking is to divert Fc gamma RI-IgG complexes from the recycling pathway, causing their retention and subsequent degradation within the cell. In contrast to Fc gamma RII-mediated endocytosis, intracellular accumulation of cross-linked Fc gamma RI-IgG complexes is not sensitive to inhibition by genistein, suggesting that the process is independent of tyrosine kinase activity.

Human high-affinity Fc IgG receptor (Fc gamma RI)-mediated phagocytosis and pinocytosis in COS cells.

The high-affinity receptor (Fc gamma RI) for the constant (Fc) portion of immunoglobulin G (IgG) is one of three Fc IgG receptor classes (Fc gamma Rs) found on mononuclear phagocytes. The functional specialization of each of the Fc gamma R classes is not well understood. Previous studies utilizing anti-Fc gamma R monoclonal antibodies (mAbs) as opsonins suggest that Fc gamma RI, like the other Fc gamma Rs expressed by macrophages, is able to mediate phagocytosis. The ability of Fc gamma RI to mediate pinocytosis, however, had not been certain, since it binds, but does not mediate, internalization of monomeric IgG in the monocytoid U937 cells. We studied Fc gamma RI-mediated internalization by introducing it into the Fc gamma R-negative fibroblastic COS cells. We found, using electron microscopy and fluorescence microscopy, that COS cells expressing Fc gamma RI are able to phagocytose IgG-coated zymosan particles and sheep red blood cells (SRBC), as well as pinocytose cross-linked IgG. There was no intracellular accumulation of monomeric IgG. Chimeric receptors which retain the extracellular domains of Fc gamma RI but lack the entire wild-type transmembrane and intracellular regions of the receptor mediated both phagocytosis and pinocytosis with equal or increased efficiency when compared to the wild-type receptor. Control COS cells transfected with CD2 rosetted, but did not phagocytose, SRBC. Attachment of phagocytic targets to COS cells is therefore not sufficient for phagocytosis. Taken together, this suggests that the extracellular domain of Fc gamma RI is sufficient for it to mediate phagocytosis and pinocytosis in this system.

Development of myotomal cells in Xenopus laevis larvae.

Electron microscopic appearances of Xenopus laevis axial myotome cells were examined through Stages 22-41 (Nieuwkoop & Faber, 1956). Differentiated contractile structures were not observed in myotome cells at or before Stage 22. At Stages 24-25, myofibrils appeared both as disordered strands in association with tubular membranes, and in different degrees of assembly into ordered sarcomeres. By Stage 28, all contractile organelles observed were organised into sarcomeres. The latter extended the length of each myocyte by Stage 35. Myofibrils were initially laid down adjacent to the cell membrane in each myocyte close to its lateral surface. They filled most of each cell by Stage 41. Membrane structures known to be associated with contractile activation in adult muscle appeared early in development. Transverse tubular, and sarcoplasmic reticular membranes, and 'triad' complexes could be demonstrated from the outset of sarcomere formation at Stage 24. In places, establishment of a regular repeating tubular system appeared to precede myofilament organisation. Examination of sections stained with ruthenium red during preparation suggested that tubular and surface membranes were continuous even at Stage 24 and all subsequent developmental stages studied here. Earlier work (Huang, 1986) has suggested that excitation-contraction coupling in embryonic muscle involves release of stored calcium in response to tubular voltage changes as in adult muscle. These findings corroborate such physiological observations.

The histochemical localization of acrosin in guinea-pig sperm after the acrosome reaction.

The protease acrosin is widely considered to be an essential component of a zona lysin which enables sperm to penetrate the zona pellucida of the egg. Sperm form a characteristic penetration slit little wider than the sperm head itself and this has long suggested that any zona lysin is attached to the sperm surface after an acrosome reaction. This paper provides the first ultrastructural evidence that this is the case. The protein acrosin inhibitor, Kunitz soybean trypsin inhibitor, has been covalently attached to the electron-dense marker, ferritin, and the conjugate incubated with guinea-pig sperm which have undergone an A23187-induced acrosome reaction. Electron microscopy shows that ferritin is distributed unevenly over the outer surface of the newly exposed inner acrosomal membrane but does not extend to the equatorial segment. This is further evidence that acrosin can be considered as a candidate for the role of zona lysin. The mechanism of sperm penetration of the zona is discussed in the light of these observations.