Actin-like filaments and membrane rearrangement in oxyntic cells ( heavy meromyosin / membrane translocation / membrane-associated filaments / HCl secretion / electron microscopy )

The secretory pole of vertebrate oxyntic cells possesses two distinct membrane systems: the apical plasma membrane which presents numerous infoldings, microvilli and processes, and a complex tubulovesicular system located in close proximity to the plasma membrane. These two membrane systems are generally believed to be interconvertible in relation to the functional state of the cell. To determine the role that filaments may play in the interconversion process, the secretory pole of rat and toadoxyntic cells was examined by electron microscopy under conditions designed to demonstrate filamentous structures, i.e., slight cellular swelling and incubation with heavy meromyosin. Filaments 50-80 A in diameter are present in close association with the plasma membrane to which they are connected by regularl spaced bridges. Heavy meromyosin-treated material reveals "decorated" filaments in topographically corresponding locations. No filaments are seen in association with membranes of the tubulovesicular system. These findings suggest that association with actin-like filaments is a step in the translocation of membranes from the tubulovesicular system to the plasma membrane. The extensive membrane rearrangements of the apical pole that accompany the transition from resting state of HUl-secreting state are a common feature of all vertebrate oxyntic cells. In the resting state, the plasma membrane at the secretory pole presents an uncomplicated topography with few projections into the lumen. Beneath the apical membrane, a very well-developed tubulovesicular system is present. The onset of HC1 secretion is accompanied by the appearance of complex infoldings and processes which increase the area of the plasma membrane at the apical zone approximately 10-fold (1). At the same time the total area of the membranes that compose the tubulovesicular system is greatly decreased. Extensive work done to clarify the relationship existing between plasma membrane and tubulovesicular system has yielded the following pertinent data: (i) morphometric studies show that the sum of the areas of apical and tubulovesicular membranes is approximately constant for all functional states of the cell (1, 2). (fi) A definite structural similarity exists between the apical plasma membrane and the membranes of the tubulovesicular system (3). (Mii) Bicarbonate-activated ATPase activity can be cytochemically demonstrated in relation to the tubulovesicular system in the resting state, while during activity it is found in relation to the plasma membrane of the apical processes (4). (iv) Inhibition of protein synthesis does not inhibit the increase of apical membrane area at the onset of secretion (5) which indicates that newly-synthesized membrane components cannot account for this increase. These data support the widely held opinion that the two nMembrane systems of the apical pole are interconvertible. An explanation of the mechanism by which changes at the initial stage of secretion take place should include, among others, at least two events which may probably occur in such an interconvertible system. The first is the transfer of elements of the tubulovesicular system toward the plasma membrane; the second is the incorporation of these elements into the plasma membrane by fusion. Up to the present, attention has been focused almost exclusively upon the membranes themselves. Little consideration has been given to the idea that such extensive changes and rearrangements at the plasma membrane level must, of necessity, entail changes of nonmembrane structures present in the cytoplasm that comes into contact with the membrane systems. Our work has been directed to the study and characterization of cytoplasmic elements that may participate in the changes that accompany secretion. In this report we describe a system of actin-like filaments that are closely associated with the plasma membrane of the secretory pole of oxyntic cells; these filaments are not associated with membranes of the tubulovesicular system. MATERIALS AND METHODS Myosin was prepared from rabbit skeletal muscle as described by Szent-Gy6rgyi (6). After several precipitation and solubilization cycles a 1% solution in 0.6 M KCI was digested with trypsin (Mann Research Laboratories, Inc., specific activity 6600 a-N-benzoyl-L-arginine ethyl ester units/mg) by the method of Lowey and Holtzer (7). The digestion was stopped with lima bean trypsin inhibitor (Mann Research Laboratories, Inc.). After dialysis against 1 mM phosphate buffer at pH 7.0, for 3 hr, the digest was centrifuged at 30,000 X g for 10 min. The supernatant containing predominantly heavy meromyosin (HMM), without further purification, was employed for the treatment of glycerinated cells. Gastric gland cell suspensions were prepared from albino rat stomachs by a procedure modified from Romrell et al. (8). The modifications introduced were: (a) crude papain (BDH Chemicals Ltd., Poole, England) at a concentration of 3 mg/ml was used instead of Pronase; no activator was added to the papain solution and (b) no DNase treatment of the initial cell suspension was carried out. This method produces a mixed suspension of gastric gland cells which are 95-98% viable as judged by trypan blue exclusion tests. After washing to eliminate papain, some cells were directly fixed and processed for electron microscopy; others were glycerinated as indicated below. The method described above was not effective in obtaining similar cell suspensions from the gastric glands of the toad Bufo 8pinulosus. In this animal, the surface epithelium of the mucosa is extremely resistant to dissociation by proteases. This prompted us to adopt a different procedure. Tissue slices (200 ,m thick) were obtained by means of a Smith-Farquhar tissue sectioner from freshly excised gastric mucosae. After glycerination, carried out as described below, part of the material was processed for electron microscopy while the rest was treated with HMM. 4032 Abbreviations: HMM, heavy meromyosin; SSB, 100 mM KC1, 5 mM MgCl2, 300 mM sucrose, 600 mM phosphate buffer at pH 7.0. Proc. Natl. Acad. Sci. USA 73 (1976) 4033 For glycerination, the material was placed in a salt-sucrose-buffer solution (SSB: KCI, 100 mM; Mg9l2, 5 mM; sucrose, 300 mM; 6 mM phosphate buffer at pH 7.0). Glycerol concentration was continuously increased by addition of a 300 mM solution of sucrose in glycerol by means of a gradient maker until the initial volume of SSB had doubled. The passage from 0 to 50% glycerol was completed in 40 min. After 10 min in 50% glycerol, SSB was added stepwise throughout a 30 min period until the concentration of glycerol was 25%. The material was then collected by centrifugation, washed in SSB to remove all glycerol, and incubated at 40 in a 0.4% solution of HMM in 1 mM phosphate buffer at pH 7.0, for 2 hr. The material was subsequently, fixed in 2% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) containing 0.1 M sucrose. After washing, the sample was postfixed in OsO4, block-stained in uranyl acetate, dehydrated in acetone, and embedded in Epon. The fine structure of the secretory pole of oxyntic cells was also studied on material which had not undergone glycerolHMM treatment. This material was obtained and treated by one of the following sequences: (a) aliquots of the gastric gland cell suspensions, obtained as described above, were fixed in 1.6% glutaraldehyde in 0.2 M sodium cacodylate buffer at pH 7.2, postfixed in OS04, dehydrated in acetone, and embedded in Epon; (b) toad gastric mucosae were excised after 45 min of histamine stimulation (10 mg/kg of histamine phosphate injected into the dorsal lymph sac); and (c) toad gastric mucosae were quickly removed from the pithed animals and incubated in vitro for 1 hr in the solution described by Villegas et al. (9) to which 60 ,M histamine had been added. Small blocks of tissue from the mucosae obtained in (b) and (c) were prepared for electron microscopy in the manner described for the cell suspensions. Silver to gray sections were obtained with diamond knives; they were stained with uranyl acetate in methanol (10) and lead citrate.