Multiple effects of Na+ removal on pancreatic secretion in vitro

Summary The effects of removing Na⁺ from the incubation medium on basal and secretagogue induced zymogen release by pancreatic fragments and isolated pancreatic acini were studied by both morphological evaluation and measurement of amylase release. In both fragments and isolated acini, removal of Na⁺ led to an increased basal secretion of zymogen granule contents from acinar cells via exocytosis; secretory material, however, accumulated in acinar and ductular lumina as a result of the lack of fluid secretion necessary to wash out the enzymes. In studies with fragments, after Na⁺ removal there was no significant increase in amylase release into the medium; isolated acini, in contrast, showed an increased amylase release consistent with the shorter distance from the acinar lumen to the bathing medium. Stimulation with either bethanechol or caerulein led to a further depletion of zymogen granules in both preparations; in the absence of Na⁺ secretory product accumulated in intracellular lakes as well as in duct lumens. The hypothesis that Na⁺ influx is important in stimulus-secretion coupling to release intracellular Ca²⁺ was directly tested by measuring ⁴⁵Ca²⁺ efflux. No effect of removing Na⁺ on ⁴⁵Ca²⁺ efflux was seen. It was concluded, therefore, that while Na⁺ is essential for pancreatic fluid secretion, it is not necessary for the secretion of zymogen granule contents into acinar lumina.Supported by NIH grant GM-19998 from the United States Public Health Service     

The differential effects of cytochalasin B on microfilament populations and cytoplasmic streaming

Summary Two distinguishable populations of microfilaments (mfs) can be identified in the radish root hair. Bundles of mfs are found throughout the cytoplasm, excluding the tip region of the hair. Single mfs occur only as a cortical array, specifically associated with the microtubules. Both mf populations are oriented parallel to the direction of streaming. Hairs grown in 5 g/ml cytochalasin B (CB) exhibit site-specific differential responses to the drug in both their streaming pattern and sensitivity of their mfs. Cytochalasin B elicits the following responses: 1. cytoplasmic streaming is reduced in all regions of the hair; 2. small particles (<1 m in diameter) still stream, whereas large particles (>1 m in diameter) no longer stream but exhibit an oscillatory or rotational motion; 3. filament bundles show increasing sensitivity to CB along the length of the hair; 4. single mfs show decreasing sensitivity to CB along the hair length. The effects of CB on cytoplasmic streaming can be related to its effects on both mf populations, thus suggesting that although mf bundles are probably involved in streaming in the sub apical and basal regions of the hair, single mfs are most likely involved in generating the slower, more irregular streaming patterns exhibited in the hair tip and CB-treated hair base.     

Effects of high concentrations of secretagogues on the morphology and secretory activity of the pancreas: A role for microfilaments

Summary Cholecystokinin (CCK) and acetylcholine, at concentrations greater than those required for maximal pancreatic enzyme secretion, elicit a submaximal secretory response. The mechanism for this secretagogue-induced unresponsiveness is unknown. Using isolated pancreatic acini of the mouse, we now find that high concentrations of secretagogues also induce a profound alteration in acinar morphology, characterized by the formation of spherical protrusions on the basal surface of the cells. Since both the determination of cell shape and exocytosis may involve calcium and contractile proteins, we used a calcium-free medium and cytochalasin B (CB) to evaluate the importance of a contractile mechanism in the secretory and morphological effects of high concentrations of CCK-octapeptide (CCK₈). Incubation in a calcium-free medium partially blocked CCK-induced unresponsiveness, but brought about dissociation of the acini. CB at a concentration of 3 g/ml caused the disappearance of apical microfilaments and, most strikingly, completely prevented the morphological alteration induced by CCK₈. Furthermore, CB converted the biphasic dose-response curve for CCK₈-induced amylase release to a monophasic shape, such that the amylase release stimulated by a high concentration of CCK₈ (10 nM) was augmented. It is concluded, therefore, that a contractile process involving microfilaments may mediate secretagogue-induced unresponsiveness in pancreatic acinar cells.     

Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes     

Effects of cytochalasin B on the cleavage furrow in mouse blastomeres

Summary Blastomeres isolated from two-cell mouse embryos were cultured until they started to cleave. When the cleavage furrow developed they were subjected to cytochalasin B (CB) and were studied with the electron microscope. The initial response to CB is that the furrow is more folded and microvillous than in the control. Later the blastomeres round up. The protrusions covered with abundant long microvilli are found scattered within their equatorial surface. Extraction with glycerol solution before fixation permits visualization of condensations of felt-like filamentous material in contact with the cleavage furrow during the initial response to CB and in the protrusions of rounded cells. We consider clumping of filaments in surface protrusions to be a specific response to CB treatment of the contractile ring.Some of the previous papers by this author have been published under the name Opas     

Scanning electron microscopy of dissociated pancreatic acinar cell surfaces

Summary The pancreatic acinar cell surfaces have been studied by SEM with a dissection technique and correlated with results obtained by TEM. The SEM results demonstrate characteristic arrangement of microplicae which in some areas are densely packed.In many areas, the microplicae are distributed in such a manner that they create zones with typical geometrical shapes and show a relatively smooth surface.These smooth areas may coincide, as indicated by correlated TEM results, with the limits of intimate contact between adjacent acinar cells which, in turn, represent part of the junctional complex. Another aspect revealed by these SEM preparations concerns the presence of groups of densely packed microplicae, arranged in regular rows and distributed along some grooves and/or infoldings of the cellular surface. On the basis of SEM and TEM information, it is likely that these structures correspond to intercellular (and possibly, in some cases, intracellular) canaliculi which topographically form a kind of extensive microlabyrinthine arrangement running along all the cell sides.One final point revealed by fractured samples concerns the finding of spherical zymogen droplets within the vesicles of the Golgi complex. Because in many scanning images these vesicles appear connected by small openings, it is suggested that they may represent a system of intercommunicating chambers (vacuoles) through which the zymogen droplets can be continuously accumulated and discharged into the acinar lumen.     

Effects of phalloidin and cytochalasin B on cytoskeletal structures in cultured rat hepatocytes

Summary In short-term cultures of rat hepatocytes, bile canaliculi enclosed between unseparated cell couplets are able to perform periodical contractions resulting in expulsion of bile. Pericanalicular cytoskeletal proteins are involved in canalicular contractility: F-actin, myosin and tropomyosin are associated around bile canaliculi, as revealed by staining with tetramethylrhodaminyl-phalloidin and by immunofluorescence. Bile canalicular contractility is distributed by cholestatic agents that are known to interfere with actin polymerization; e.g., phalloidin and also cytochalasin B inhibit canalicular contractility and cause pericanalicular vacuolization and formation of blebs. Whereas the association of the cytoskeletal proteins is not affected by treatment with cytochalasin B, treatment with phalloidin results in dissociation of F-actin and myosin, indicating that binding of phalloidin to F-actin impairs its molecular interaction with myosin.     

The effect of cytochalasin B on the enucleation of erythroid cells in vitro

Summary The process of nuclear extrusion continues when erythroid cells of mouse spleen are placed in vitro, although the process may be considerably retarded. This allows visualization of unusually large numbers of enucleating cells after 30 min in vitro. With this system, the mechanism of enucleation can be analyzed both qualitatively and quantitatively. We have used this system to study the effects of cytochalasin B and have found it to be a potent inhibitor of enucleation. These results suggest that microfilaments are involved in the process of enucleation.Supported by NSF Grant PCM 78-00573     

Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells

We have tested the effects of alcohols differing by their alkyl chain length on the membrane channels and amylase secretion of rat pancreatic acinar cells. In intact acini, alcohols with a chain of seven, eight, or nine carbons (C-7, C-8, and C-9) induced dye uncoupling and increased basal amylase release. These effects were readily reversible after alcohol removal. By contrast, an alcohol with a chain of 15 carbons (C-15) and several alcohols with chains of fewer than six carbons (C-2, C-4, and C-6) did not uncouple acinar cells and had no effects of amylase secretion. Neither did alkanes and oxidized derivatives of C-7 and C-8 alcohols did not affect dye coupling. Double patch-clamp experiments on pairs of acinar cells, under conditions of strong cytosolic Ca2+ and pH buffering, showed that C-7, C-8, and C-9 alcohols blocked completely and reversibly the electrical conductance of junctional channels. Furthermore, studies of single voltage-clamped acinar cells revealed that the uncoupling alcohols did not affect the resting nonjunctional membrane conductances. Thus the alcohols that did not affect acinar cells coupling did not affect amylase secretion, whereas the alcohols that caused uncoupling increased secretion. The latter effect was not mediated by changes in the conductance of nonjunctional membrane, cytosolic Ca2+, and pH and, as revealed by an immunological hemolytic plaque assay for amylase, had a time course consistent with the rapid (within 1 min) inhibition of coupling. These data provide new support for the view that the regulation of cell-to-cell communications is correlated with that of digestive enzyme secretion.     

An analysis of the role of actin during pollen activation leading to germination in pear (Pyrus communis L.): treatment with cytochalasin D

Summary Major stages of actin organization during activation leading to germination of pear (Pyrus communis L.) pollen were disrupted by treatment with 5 g/ml cytochalasin D (CD), and the effects of the drug were monitored with rhodamine-phalloidin staining. CD induced the formation of granules or short rods in the place of the filamentous arrays that occur in normally developing pollen. Filamentous arrays, however, returned upon removal of CD. Pollen incubated directly in CD showed a gradual disappearance of circular actin profiles and their replacement by either granules or, less frequently, short rods. These granules and rods initially had a random distribution in the cell, but with time in CD they became localized at one of the three germination apertures. Pollen was also allowed to reach three stages of microfilament (MF) organization (initial fibrillar arrays, interapertural MFs, and MFs confined beneath a single aperture) prior to being continously exposed to CD. After CD treatment, germination was blocked and the number of cells containing short rods increased, but movement of actin to a single aperture continued. Finally, when pollen at different stages of MF organization was treated with a CD pulse and then transferred to drug-free medium, germination was delayed regardless of the stage of MF organization at the time of treatment. The results indicate that an uninterrupted progression of actin organization is essential for pollen germination, but that movement of actin in the cell is CD-insensitive.     

The influence of concanavalin A and cytochalasin B on pinocytotic activity inAmoeba proteus

Summary Addition of concanavalin A to a suspension ofAmoeba proteus brings about cellular agglutination and the formation of what appear to be pinocytotic channels in cell surface projections. Although concanavalin A apparently brings about pinocytotic channel formation, it does not elicit bulk medium uptake or surface membrane turnover. Cytochalasin B brings about an initial cessation of locomotion and the development of a number of randomly distributed pseudopods. After a 30 to 45 minute exposure to cytochalasin B, the cells resume their normal appearance and pattern of locomotion. Cytochalasin B itself has no influence on inducing pinocytotic channel formation or membrane turnover, but when pinocytosis is induced with 0.01% alcian blue, pinocytotic activity is greatly intensified by the presence of cytochalasin B.     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis. Dedicated to Professor P. Heinrich on the occasion of his 60th birthday     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis.     

Effect of cytochalasin D on secretion by rat pancreatic acini

Using the model of isolated acini the effect of cytochalasin D (CD) on rat pancreatic secretion in vitro was studied. The influence of CD (0.01-10 micrograms/ml = 0.02-19.7 microM) on amylase secretion and 3H-pulse-labelled protein release was measured under two sets of conditions: (a) basal, and (b) stimulated by 77pM caerulein. Basal secretion was not altered, but stimulated secretion of amylase and 3H-labelled proteins were similarly inhibited by up to 45%. It is concluded that CD affects only exocytosis of zymogen granules and not intracellular transport.     

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.     

Low extracellular pH induces damage in the pancreatic acinar cell by enhancing calcium signaling

Low extracellular pH (pHe) occurs in a number of clinical conditions and sensitizes to the development of pancreatitis. The mechanisms responsible for this sensitization are unknown. Because abnormal Ca(2+) signaling underlies many of the early steps in the pathogenesis of pancreatitis, we evaluated the effect of decreasing pHe from 7.4 to 7.0 on Ca(2+) signals in the acinar cell. Low pHe significantly increased the amplitude of cerulein-induced Ca(2+) signals. The enhancement in amplitude was localized to the basolateral region of the acinar cell and was reduced by pretreatment with ryanodine receptor (RYR) inhibitors. Because basolateral RYRs also have been implicated in the pathogenesis of pancreatitis, we evaluated the effects of RYR inhibitors on pancreatitis responses in acidic conditions. RYR inhibitors significantly reduced the sensitizing effects of low pHe on zymogen activation and cellular injury. These findings suggest that enhanced RYR-mediated Ca(2+) signaling in the basolateral region of the acinar cell is responsible for the injurious effects of low pHe on the exocrine pancreas.     

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.