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Abstract. The major part of mast cell actin is Tritonsoluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, though filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be ~2.5/~g/106 cells (cytosolic concentration ~110 #M.After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, *60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP--y-S together with Ca 2÷. In the presence of cytochalasin E the requirement for Ca 2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by CaE+-dependent actin regulatory proteins; their presence is indicated by a Ca2÷-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca 2+ and cause variable extents of CaE+-independent secretion.Exposing the permeabilized cells to increasing concentrations of Ca 2÷ caused a progressive decrease in F-actin levels as measured by flow cytometry of RPstained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP--y-S was not required for the Ca2+-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur. p ERMEABILIZED rat mast cells provide an excellent system for investigating the mechanism of exocytosis. In response to provision of Ca 2÷ and GTP (or its analogues), they release up to 100% of their contained histamine and hexosaminidase, both of which can be rapidly and sensitively measured (16). The exocytotic process, which may be studied by providing appropriate effect.ors (bypassing the receptor-agonist interaction), involves fusion of the granular and plasma membranes followed by the release of granule contents. Since all mammalian cells contain an actin-rich network in the cortical region (6), it has been suggested that disassembly of the cortical cytoskeleton might be a prerequisite for exocytosis (3,7,26,42). On the other hand, the motile elements of the cytoskeleton are also thought to be involved in the transport of vesicles to the plasma membrane. Several studies have demonstrated increased actin filament assembly after cell stimulation (10,39,41). Controversial conclusions have also been presented on the basis of experiments involving the use of microfilament-specific agents such as phalloidin and cytochalasin (2,13,16,19,29,30,34,42,44). Thus, the exact role (if any) of the cytoskeleton in the...

SUMMARY1. The studies described herein involve the use of light scattering measurements to characterize the ultrastructural arrangement of the constituent collagen fibrils in rabbit corneal stromas.2. Theoretical light scattering techniques for calculating the scattering to be expected from the structures revealed by electron micrographs are discussed, and comparison with the experimental light scattering tests the validity of these structures.3. The wave-length dependence of light transmission and of angular light scattering from normal corneas is in agreement with the short range ordering of collagen fibrils depicted in electron micrographs.4. The transmission measurements on oedematous rabbit corneas indicate that transmission decreases linearly with the ratio of thickness to normal thickness.5. The wave-length dependence of transmission through cold swollen corneas indicates that the increased scattering is caused by large inhomogeneities in the ultrastructure. Electron micrographs do, indeed, reveal the presence of such inhomogeneities in the form of large regions completely devoid of fibrils.

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage.clamp measurements using the patch-clamp technique, and (c) by measurements of exocytos~is in response to entry of Ca 2+ and GTP')'S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 #M. In normal saline containing 1 mM MgCl~ and 2 mM CaCl~, dye uptake and electric conductance are detectable at 100 #M ATP corresponding to 4 #M ATP 4-. The permeabilization is half-maximal at an ATP 4-concentration of 5-20 #M with a Hill coefficient near 2. The ATP-induced whole.cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant <65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of ~200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of ~40 pS and the single-channel conductance obtained from noise analysis is ~2-10 pS. Entry of Ca 2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP~S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

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