Effects of colchicine and vinblastine on in vitro gastric secretion DINKAR K. KASBEKAR AND GILAD S. GORDON Department of Physiology and Biophysics, Georgetown School of Medicine, Washington, D. C. 20007
KASBEKAR,
DINKAR
K.,
AND
S.
GILAD
Effects uf
GORDON.
colchicine and uinblastine on in vitro gastric secretion. Am. J. Physiol. 236(5): EGO-E555,1979 or Am, J, Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(5): E550-E555, 1979.-The effects of colchicine and vinblastine on in vitro bullfkog gastric mucosal preparations were studied with respect to H+ and pepsinogen secretion, In the concentration range of l-50 mM, an initial but transient colchicine-mediated stimulation of Hf secretion is followed by a dose-dependent inhibition. The transient stimulation of H’ secretion can be confirmed in resting preparations in the absence of added secretagogues. Xn the same concentration range, colchicine inhibits pepsinogen secretion to a greater degree than Hf secretion. Vinblastine (10B5-5 x 10m4 M) was more effective than colchicine in inhibiting both H’ and pepsinogen secretion. The kinetics of inhibition of secretion by both colchicine and vinblastine were slow. Cytochalasin I3 had no effect on either secretion. gastric H’ secretion; ing mucosa
EVIDENCE
HAS
pepsinogen
ACCUMULATED
secretion;
in
reCent
cytochalasin
years
8; rest-
Supporting
the involvement of smooth endoplasmic reticulum, tubulovesicular elements, and microvilli in the gastric acid secretory process (5, 10, 23). In the amphibian gastric mucosa, electron-microscopic studies indicate that the smooth apical surface of the tubular cell becomes elaborated with microvilli on stimulation of acid secretion with a concomitant reduction in the cytoplasmic tubulovesicular elements. These changes are accompanied by an increased turnover of the polar moieties of the mucosal membrane phospholipids (13). Similar observations have been made in the mammalian gastric epithelia where the canalicular surface of the parietal cell undergoes major alterations on stimulation of acid secretion. Relatively little is known, however, about the events responsible for the orderely transformation of these elements in the postulated (‘secretory apparatus” of the acid-secreting cell. Studies with a number of secretory tissues have implicated microtubules and microfilaments in the repetitive assembly, disassembly, and reassembly of the various structural elements involved in the secretion process. These cytoskeletal elements appear to be associated with several types of intracellular movements (3) including chromosome movement (9), transport and release of secretory products (15, 19, 20, 25, 27), and cytoplasmic streaming (18). The apical surface transformations in the E550
University
acid-secreting cell invoked by stimulation or inhibition of gastric H+ secretion appear to undergo somewhat similar movements. The antimitotic agents colchicine and vinblastine and others are known to exert disruptive effects on microtubules in vivo (9, 16) and to interact with microtubule subunit protein in vitro (2, 28). These agents have been employed extensively as tools in the study of microtubules in cellular functions. Cytochalasin B has been reported to disrupt microfilaments in several cell types (17) and has been shown to interact with actin (24). In an attempt to determine whether microtubules or microfilaments participate in the sequence of events leading to acid or pepsinogen secretion, we have studied the effects of these agents on H+ and pepsinogen secretions by the isolated frog gastric mucosal preparations. MATERIALS
AND
METHODS
Adult male frogs (Rana catesbeiam) were obtained from Mogul ED, Oshkoh, WI, and maintained unfed in running tap water for at least I wk before study. Colchitine was a product of Calbiochem, San Diego, CA; vinblastine and cytochalasin B were obtained from Sigma Chemical Co., St. Louis, MO. Hemoglobin (enzyme substrate powder) was supplied by Grand Island Biological Company, Grand Island, NY, and a 5% suspension of the powder was dialyzed extensively against 1.0 mM HCl at 4OC before use in pepsinogen assays. Burimamide was a generous gift from Dr. M. E. Parsons of Smith Kline & French Laboratories, London, England. All other chemicals were the highest grade purity available. Acid and pepsinogen secretion was monitored in isolated gastric epithelia mounted on polyethylene tubes. When electrical parameters were measured, chambered mucosal preparations were used. Briefly, the frog was pithed, the stomach removed and slit along its lesser curvature, and the mucosa separated from the smooth muscle coat by blunt dissection. The mucosa was divided longitudinally into approximately equal halves and one half was used as an appropriate control. Care was generally exercised to exclude esophageal and pyloric regions from the effective tissue area used in the study. The serosal and luminal bathing solutions were comprised of the frog Ringer solution (13) and 120 mM unbuffered NaCl, respectively, and both solutions were gassed with 95% 02-5s COZ. Acid secretion was measured with the pH stat method (4) at the end-point setting of 4.5 with 10 mM NaOH used for titration. Transepithelial poten-
0363-6100/79/0000-0000$01.25
Copyright 0 1979 the American Physiological Society
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COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E551
SECRETION
tial difference (PD) was monitored with a Kontron recorder with a pair of calomel half-cells connected to the chamber via balanced agar bridges. Resistance was calculated from the change in PD in response to passing a 2O+A current through a pair of saline agar bridges. Pepsinogen secretion was determined as pepsin activity in aliquots of luminal bathing media that were withdrawn completely every hour and replaced with fresh luminal solutions. Mucosal pepsinogen content was determined in aliquots of 10,000-g supernatant fractions of the homogenates prepared in 5 mM sodium acetate buffer, pH 4.0. Briefly, the pepsin assay procedure (I, 21) involved incubation of the enzyme with (final concentrations in 0.5 ml total volume) 2% hemoglobin in 0.1 N HCl as the substrate and 10 mM glycine buffer, pH 3.0 for 10 min at 37°C; the 0.1 N HCI used for diluting the hemoglobin buffered the incubation mixture finally at a pH of 1.8. Pepsin activity was terminated by the addition of 0.5 ml of 6% cold trichloroacetic acid (TCA). The hydrolysis products of hemoglobin contained in the filtrate as TCAsoluble tyrosine peptides were assayed by the method of Lowry et al. (14), with tyrosine used as standard. The enzyme activity was expressed as peptic units (PU), 1 PU representing an activity of pepsin that releases 0.1 pmol of tyrosine from 2% hemoglobin at pH 1.8 in 10 min at 37°C (7). The rate of secretion is expressed either as a) peptic units secreted per square centimeter per hour into the luminal bathing solution or b) the first-order rate constant for the decline of mucosal pepsinogen content. In the latter case, the rate constant was determined from the slope of the semilogarithmic plot of the pepsinogen remaining in the mucosa as a function of time. For this purpose, the mucosal pepsinogen content at zero time was calculated by adding the pepsinogen remaining in the mucosa at the end of experiment to the total amount secreted; subtracting the cumulative amounts of pepsinogen secreted at successive intervals yielded values for mucosal pepsinogen levels at the start of each interval. The expression of the pepsinogen secretion data as the first-order rate constant appear valid for the in vitro preparation, because little if any pepsinogen synthesis was observed under our experimental conditions in the absence of an amino acid supplement. This was established in preliminary experiments. Mucosae were incubated with 50 PCi of [3H]leucine (New England Nuclear, 60 Ci/mmol) in the serosal bathing medium for up to 6 h and the entire luminal solution (2-3 ml) containing secreted pepsinogen was loaded onto a Sephadex G-25 column. The column was eluted with 120 mM NaCl and 1.0~ml fractions were collected until pepsinogen appeared in the effluent, as determined by pepsin activity. No radioactive leucine was found in the fractions associated with pepsin activity, indicating that little de novo pepsinogen synthesis occurred under these experimental conditions. The relationship between pepsinogen and H+ secretion was expressed arbitrarily as a ratio, PU/H’. Resting gastric mucosal preparations were obtained as described previously (II). Various agents were added to the serosal bathing medium and, at concentrations of colchicine greater than 10 mM, isotonicity of the medium was adjusted bv a corresponding reduction in the
amounts of NaCl. All studies with colchicine out in the dark.
were carried
RESULTS
Effect of colchicine on H* secretion, PD, and resistance. Colchicine concentrations below 1 mM have no observable effects on acid or pepsinogen secretion in histamine-stimulated mucosae, At concentrations greater than I mM (IO-50 mM), there is a kinetically slow, biphasic effect on H+ secretion (Fig. 1). A transient initial stimulation of H+ secretion to a small but significant degree (l&30%) over that obtained with supramaximal histamine stimulus is followed by concentrationdependent inhibition. The inhibition can be reversed almost completely by repeated washouts of colchicine over a period of 2-3 h. In experiments in which the concentration of colchicine is increased stepwise from 0.01 to 50 mM at hourly intervals, each colchicine increment results in an initial rapid rise in PD followed by its return to base-line level over a period of 15-30 min (Fig. 2). The transmural resistance does not show significant changes until concentrations of colchicine above I mM are reached. The rise in resistance observed is consistent with the gradual decline of H+ secretion at higher colchicine concentrations. Prolonged incubation at higher colchicine concentrations eventually leads to a decline of short-circuit current, suggesting an inhibition of active chloride transport. Colchicine stimulation of H’ secretion in resting mucosa, Since the predominant effect of colchicine is to inhibit Hf secretion, it is difficult to explain the initial transient and significant stimulation. Furthermore, in most studies, as the inhibition of H+ secretion progressed at higher concentrations of colchicine, replacement of I
tSTAMlNE U4 COtCHIClNE
M
100
20 0
1
1
I
d501
4 6 8 HOURS FIG. 1. Effect of colchicine on H’ secretion, Mucosal halves from the same epithelium were stimulated with 1W4 M histamine 1 h after isolation. At the end of 2nd h, colchicine at indicated concentrations (1-50 mM) was added to one half while the second half was used as control. H+ secretion at the beginning of 3rd h was set arbitrarily to fUO% in each experiment and subsequent rates for both experimental and control halves were expressed as percentages of this rate. Insert shows dose dependence of inhibition 5 h after colchicine treatment. Vertical lines show t 1 SE for 3 or more determinations. 2
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D. K. KASBEKAR
E552 nutrient bathing medium with a fresh one containing colchicine resulted in a transient and partial reversal of inhibition. This transient stimulation of H+ secretion does not appear to be an artifact. In resting gastric mucosal preparations, colchicine has similar effects; it stimulates secretion from resting zero H+ secretion in the absence of any added secretagogues and this stimulation is followed by the usual pattern of inhibition seen in spontaneously secreting or histamine-stimulated mucosa
I
N E200 ;
‘e.*
100
-
lO-4 M HISTAMINE COLCHICINE hM)
0.010.1 I.0 p 20 A*’ ---a
l
-a-m-a
I
AND
G, S. GORDON
(Fig. 3). This stimulation may be due to the presence of a trace contaminant in colchicine, or colchicine per se may be a histamine releaser in the gastric mucosa and thus may stimulate secretion. Alternatively, colchicine may be metabolized by the mucosa and a metabolite may be responsible for the stimulatory effect. We have not pursued this aspect further and therefore have no explanation for the observed stimulation of H+ secretion by this agent. Colchicine effects on pepsinugen secretion and cumparison with H+ secretion. The fundic region of frog gastric mucosa contains and secretes pepsinogen in response to cholinergic stimuli, gastrin, and histamine (unpublished observations). The typical distribution of pepsinogen in the gastroesophageal region is shown in Fig. 4. An analysis of gastric and esophageal mucosae from five frogs yielded values of 3.4 t 0.3 and 12.1 f- 1.8 (mean t SE) PU/mg wet wt, respectively. Current evidence
4
I -
-I
‘-+-+,,,
l1 2
0
1 4
1 6
HOURS
1 0 FIG.
increasing Colchicine
2
4 I-OURS
6
3. Stimulation of resting H’ secretion by colchicine. Mucosal halves were brought to resting state by 18-h preincubation (not shown). Colchicine (10 mM) was added to one half (C) and 0.1 mM histamine (H) to the other at time indicated by arrow. In 2 other experiments colchicine-stimulated H+ secretion rates were similar and also declined faster with time compared with those obtained with corresponding histamine controls. FIG.
8
2. Typical response of PD, resistance, and H’ secretion to concentrations of colchicine in histamine-stimulated mucosa. concentrations were increased at hourly intervals as shown
1500
0 w
1000
F 3 u3
w 2 z-. 3
20 Distance
30 from
Pylorus
40
FIG. 4. Distribution of pepsinogen in gastroesophageal epithehuk Transverse strips (5 mm) were cut from the pyloric end, homogenized in acetate buffer, and assayed for pepsin activity.
50
(mm)
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COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E553
SECRET1ON
very strongly indicates that, unlike mammalian par&al cells, the frog tubular cell secretes both H+ and pepsinogen (12, 22). Since microtubules and microfilaments appear to be more intimately involved in the secretion of proteins in various tissues, we determined the effects of colchicine on pepsinogen secretion and compared the results with those on H+ secretion. The rates of H+ and pepsinogen secretion and the PU/H+ ratios for hourly intervals by the control and colchicine-treated mucosal halves are shown in Table 1 and Fig. 5, The data indicate that at 10 mM serosal colchicine concentration, pepsinogen secretion declines relatively more rapidly than H’ secretion, with a concomitant decline in PU/H+ compared with control without added colchicine. The PU/ H+ values for the control and colchicine-treated mucosal halves in four different studies were (mean t SE) 7.1 t I.0 and 1.5 & 0.3, respectively. This decline is mostly due to a decrease in the amount of pepsinogen secretion and not due to the initial small stimulation of H+ secretion observed with colchicine. These findings are in contrast to those noted with other inhibitors of Hf secretion, viz., thiocyanate or burimamide, where the decline in the rate of pepsinogen secretion lags significantly behind that of acid secretion (Fig. 6 and Table 2), resulting in increased PU/H+ ratios compared with the untreated controls. At
higher concentrations of colchicine, both H” and pepsinogen secretion rates decline rapidly, with little change in PU/H+. Effects of vinblastine on Hf andpepsinogen secretion. Vinblastine inhibition of pepsinogen and Hf secretion is shown in Fig. 7. Vinblastine (10w5-5 x 10m4M) is more effective than colchicine in inhibiting both secretions. Unlike colchicine, vinblastine does not stimulate H’ secretion transiently and the kinetics of inhibition of H+ secretion almost parallel that of pepsinogen, except at
mM 0
1 10 25 Values
are means
t4, h
k, h-’
0.124 0,117 0.092 0,046 0,035
k 1 SE for 4 or more
I
determinations.
10-4M
I.OmM
e
B’amide SCN-
~
7
5.7 * 0.2 5.9 -t- 0.4 7.5 t 0.3 15.0 -+ 0*2 20.0 -t- 0.2
50
0.1 mM Histomh
H ~20mM
TABLE 1. R&e constants for loss of mucosal pepsinogen in presence of colchicine in histamine-stimulated mucosae --.-- _ Colchicine,
I
FIG. 6. Effect of burimamide and thiocyanate on H’ rates in histamine-stimulated mucosae, Mucosal halves with 0.1 mM histamine and rates of H+ and pepsinogen observed for the next 4 h. Burimamide (1.0 mM) and mM) were then added to the individual halves and the studied for another 3 h. Note relatively slow decline secretory rates in contrast to H’ rates and compare colchicine in Fig. 5.
and pepsinogen were stimulated secretion were thiocyanate (20 secretory rates of pepsinogen with effects of
Histamine
owlthout
Colchlclnc
*with
Colchlclne
10
I
*
\
I
0 HrS-
I
I
4
8
FIG, 5. Effect of colchicine on histamine-stimulated H’ and pepsingen secretion. Mucosal halves were stimulated with histamine, and colchicine (10 mM) was added to one half while the other was used as control. Cross-hatched bars in top and bottom diagrams represent acid and pepsinogen secretion rates, refor the colchicine-treated spectively, half; open bars represent the corresponding controls. Insert shows PU/H+ for mucosal pair as a function of time. Note rapid decline of pepsinogen secretion in colchicine-treated half.
HOURS
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E554
D. K. KASBEKAR
AND
G. S. GORDON
2. PU/W ratios in histamine-stimulated mucasal halves beforeand after treatment with burimamide or tIz%ocyanate _._ _.._-------.~--.-..~--------- --- -,_._. __-.----.---- --.---- -------- W/H+ I TABLE
dependent inhibition of secretion with a concomitant rise in transmural resistance. The rate of decline of Hf secretion is also enhanced with increasing concentrations of the drug. In contrast, the decline in pepsinogen secretion is monophasic and precedes that of H’, as indicated by Before treatment After treatment decreasing PU/H’ ratios with time. Pepsinogen secretion 4h 5h 6h 7k 8h 9h is thus more sensitive to colchicine than acid secretion. Histamine, 0.1 4.9kO.8 6.3~1.0 6.ltl.I The effects of vinblastine, which also inhibits gastric mM secretion, differ in some respects from those of colchicine. + Burimamide, 36k-2 185k12 170-e-14 The concentrations required for complete inhibition of ImM both H* and pepsinogen secretion are considerably lower + Thiocyanate, 24-t-3 68-t- 7 207411 (5 x IF4 M) than those of colchicine, The initial tran20 mM --sient stimulation of H+ secretion observed with colchicine PU/H’ ratios are means t I SE for 4 experiments, similar to those is not seen with vinblastine. The decline of the H’ described in Fig. 6. Ifn each experiment, the 4th-, 5th-, and Gth-h periods served as control periods; at the end of the 6th h, one mucosal half was secretion rate with vinblastine almost parallels that of treated with burimamide and the other with thiocyanate. the pepsinogen secretion rate, so that the PU/H’ ratios do not show major changes as the inhibition of secretion \ progresses. On the other hand, the slow onset of inhibitoo tion is common to the two agents and the inhibition can be reversed in both cases by repeated washouts. The latter observation indicates that their inhibitory effect is not due to irreversible binding or damage to the tissue. Another interesting observation from the present study is the kinetics of colchicine inhibition of pepsinogen secretion vis-A-vis acid secretion in comparison with the effects of other classic inhibitors of acid secretion. Thus, thiocyanate and burimamide, which inhibit acid secretion, also inhibit pepsinogen secretion, With these agents, inhibition of pepsinogen secretion lags behind acid secretion so that there is an initial rapid increase in PU/H” as the inhibition progresses. On the other hand, as discussed above, with colchicine there is invariably a decrease in IO lC5 u4 c3 PU/H’, primarily as a result of greater decline in the VINBLASTINE (Ml rate of pepsinogen secretion. Since gastric secretogogues that stimulate H+ secretion also stimulate pepsinogen FIG. 7. inhibition of HW and pepsinogen secretory rates by vinblastine. Mucosal halves were stimulated by 10W4 M histamine, and one secretion in the frog gastric mucosa, colchicine dissociahalf was treated with vinblastine at indicated concentration while the tion of the two secretions from the same cell type may be other half was used as a control. Percent inhibition of secretion rates 5 useful in studying the point of divergence in the sequence h after vinblastine treatment was calculated as in Fig. 1. Numbers in of events leading to stimulation of the respective secreparentheses show average G.&-order rate constants (h-l) for pepsinotions by the gastric secretagogues. gen secretion. O-----O H+ secretion rate; t----o pepsinogen secretion rate. Vertical bars show I SE for 3 or more determinations. The morphological evidence for involvement of cytoskeletal elements in the gastric secretory process is, at present, merely correlative. Vial and Garrido (26) have higher concentration (5 x 1K4 M), when the rate of H’ secretion declines to zeru while pepsinogen leakage cun- demonstrated a change in the orientation of actin microtinues at a low basal rate. Like colchicine, however, the filaments with secretion and Forte and his colleagues (5) have shown the presence of microtubules in the mamkinetics of vinblastine inhibition are slow. Cytochalasin B (10-“-10S3 M) had no effect on either malian oxyntic cell just beneath the secretory surface in the apical region. They have also demonstrated cyclical pepsinogen or H+ secretion. changes in the microfilament orientation that parallel the changes in microvilli and tubulovesicular elements DISCUSSION with secretion. However, the microfilaments they deThe data presented above show that colchicine has scribe are not identified either as actin or the nonactin complex effects on gastric secretion. At concentrations filaments described in other tissues (6). A recent study greater than I mM, the drug has a biphasic action on by Hynes and Destree (8) indicates that nonactin filaacid but not on pepsinugen secretion. Within an hour of ments, although unaffected by cytochalasin B, are discolchicine addition, H” secretion increases. The stimu- rupted by colchicine. No report is made of the effects of lation, however, is transient and lasts for longer periods other agents such as vinblastine. at lower than at higher concentrations. Preliminary obThe data presented here can be considered as another servations indicate that in both the spontaneously se- line of correlative evidence supporting the contention creting and the resting mucosa this transient stimulation that cytoskeletal elements may be involved in the gastric by colchicine can be abolished by burimamide. The initial secretory response, whether they are microtubules or the increase in H’ secretion is followed by a concentrationmicrofilaments described by Hynes and Destree (8). ObDownloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 16, 2019.
COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E555
SECRETION
viously binding studies must be performed in order to target the effects of these agents. This is also necessary for determining the basis of the relatively large concentrations of colchicine needed for secretory inhibition. The evidence at hand does not differentiate between permeability, metabolic destruction, or a large pool of unpolymerized subunits. However, both the morphological and physiological studies clearly indicate that this one facet
of the gastric secretory gation. The technical assistance,of edged. This study was supported
PCM 76 09971
process
warrants
Ms. Amita by National
Desai
further is gratefully
Science
Foundation
investiacknowlGrant
l
Received
24 August
1978; accepted
in final
form
21 December
1978.
REFERENCES
1. ANSON, M. L., AND A. E. MIRSKY. The estimation 2.
3. 4.
5.
6.
7.
8. 9.
10.
11, 12.
13.
14.
15,
of pepsin with hemoglobin. J. Gen. 1Dhysiol, 16: 59-63, 1932. BORISY, G. G., AND E. W. TAYLOR. The mechanism of action of colchicine: binding of colchicine-3H to cellular protein J, CeZZ BioL. 34: 525-533,1967. BUCKLEY, I. K., AND K. R. PORTER. Cytoplasmic fibrils in living cultured cells. Protoplasma 64: 349-380, 1967. DURBIN, R. P., AND E. HEINZ. Electromotive chloride transport and gastric acid secretion in the frog. J. Gen. PhysioZ. 41: 103% 1047,1958. FORTE, T. M., T. E. MACHEN, AND J. G. FORTE. Ultrastructural changes in oxyntic cells associated with secretory function. a membrane recycling hypothesis. GastroenteroZogy 73: 941-955, 1977. GOLDMAN, R. D., AND D. M. KNIPE. Function of cytoplasmic fibers in non-muscle cells. CoZd Spring Harbor Symp Quant. BioL 37: 523-534, 1972. HPRSCHOWITZ, B. I. Secretion of pepsinogen. In: Handbook of Physiology. Alimentary CanaL Washington, DC.: Am. Physiol. Sot. 1968, sect. 6, vol. II, p, 889-918. HYNES, R. O., AND A. T. DESTREE. Ten nm filments in normal and transformed celIs. Cell 13: 151-163, 1978. INOUI?, S., AND H. SATO. Cell motility by labile association of molecules: the nature of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 50, Suppl.: 259-292,1967. ITO, S., AND G. C. SCHOFIELD. Studies on the depletion and accumulation of microvihi and changes in the tubulovesicular compartment of mouse parietal. cells in relation to gastric acid secretion, J. CeZZ Biol, 63: 364-382, 1974. KASBEKAR, D. K. Studies of resting isolated frog gastric mucosa. Proc. Sot. Exp. Biol. Med. 125: 267-271, 1967, KASBEKAR, D. K., AND G. H. BLUMENTHAL. Isoltion, culture and some properties of frog gastric tubular cells. Gastroenterobgy 73: 881,1977. KASBEKAR, D. K., G. M. FORTE, AND J. G. FORTE. Phospholipid turnover and ultrastructural. changes in resting and secreting bullfrog gastric mucosa. Biochim. Biophys. Acta 163: l-13, 1968. LOWRY, 0. H., N. J. ROSEBROUGH, A, L. FARR, AND R, J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. &em. 193: 265-275,19X MALAISSE, W. J., F. MALAISSE-LAGAE, E. VAN OBBERGHEN, G. SOMERS, G. DAVIS, AND L. ORCI. The microtubular microffiamentous system of the pancreatic P-cell. Excerpta Med. Int, Congr,
Ser. 273: 282-287, 1973. 16. MALAWISTA, S. E., H. SATO, AND K, G. BENSCH. Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science 160: 770-772,1968. 17. MCGUIRE, J., AND G. MOELLMAN. Cytochalasin B: effects on microfilaments and movement of melanin granules within melanocytes. Science 175: 642-644, 1972. 18. NACHMIAS, V. T., H. E. HUXLEY, AND D. KESSLER. Electron microscope observations on actomyosin and actin preparations from Physarum polycephalum, and their interaction with heavy meromyosin subfragment I from muscle myosin. J. MoL BioZ. 50: 83-90, 1970. 19. ORR, T. S. C., D. E. HALL, AND A. C. ALLISON. Role of contractile microfilaments in the release of histamine from mast cells. Nature London 236: 350-351, 1972. 20. PAULSON, J. C., AND W. 0. MCCLURE. Inhibition of axoplasmic transport by colchicine, podophyllotoxin, and vinblastine: an effect on microtubules. Ann. NY Acad. Sci. 253: 517-527, 1975. 21. SCHLAMOWITZ, M., AND L. U. PETERSON. Studies on the optimum pH for the action of pepsin on “native” and denatured bovine serum albumin and bovine hemoglobin. J. BioZ. Chem. 234: 31373145, 1959. 22. SEDAR, A. W. Electron microscopy of the oxyntic cell in the gastric glands of the bullfrog (Rana catesbeiana). J. Biophys. Biochem. Cytol. 9: I-18, 1961. 23. SEDAR, A. W. Fine structure of the stimulated oxyntic cell. Federatiolz Proc. 24: 1360-1367, 1965. 24. SPUDIK, J. A., AND S. LIN. Cytochalasin B, its interaction with actin and actomyosin from muscle. Proc. NatZ. Acad. Sci. USA 69: 442-446, 1972. 25. THOA, N. B., G. F. WOOTEN, J. AXELROD, AND I. J. KOPIN. Inhibition of release of dopamine P-hydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine or cytochalasin 13. Proc. NatL Acad. Sci. USA 69: 520-522, 1972. 26. VIAL, J. D., AND J. GARRIDO. Actin-like filaments and membrane rearrangement in oxyntic cells. Proc. NatZ. Acad. Sci. USA 73: 4032-4076, 1976. 27. WILLIAMS, J. A., AND J, WOLFF. Possible role of microtubules in thyroid secretion. Proc. NatZ. Acad. Sci. USA 67: 1901-1908, 1970. 28. WILSON, L. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyhotoxin. Biochemistry 9: 4999-5007, 1970.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 16, 2019.
KASBEKAR,
DINKAR
K.,
AND
S.
GILAD
Effects uf
GORDON.
colchicine and uinblastine on in vitro gastric secretion. Am. J. Physiol. 236(5): EGO-E555,1979 or Am, J, Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(5): E550-E555, 1979.-The effects of colchicine and vinblastine on in vitro bullfkog gastric mucosal preparations were studied with respect to H+ and pepsinogen secretion, In the concentration range of l-50 mM, an initial but transient colchicine-mediated stimulation of Hf secretion is followed by a dose-dependent inhibition. The transient stimulation of H’ secretion can be confirmed in resting preparations in the absence of added secretagogues. Xn the same concentration range, colchicine inhibits pepsinogen secretion to a greater degree than Hf secretion. Vinblastine (10B5-5 x 10m4 M) was more effective than colchicine in inhibiting both H’ and pepsinogen secretion. The kinetics of inhibition of secretion by both colchicine and vinblastine were slow. Cytochalasin I3 had no effect on either secretion. gastric H’ secretion; ing mucosa
EVIDENCE
HAS
pepsinogen
ACCUMULATED
secretion;
in
reCent
cytochalasin
years
8; rest-
Supporting
the involvement of smooth endoplasmic reticulum, tubulovesicular elements, and microvilli in the gastric acid secretory process (5, 10, 23). In the amphibian gastric mucosa, electron-microscopic studies indicate that the smooth apical surface of the tubular cell becomes elaborated with microvilli on stimulation of acid secretion with a concomitant reduction in the cytoplasmic tubulovesicular elements. These changes are accompanied by an increased turnover of the polar moieties of the mucosal membrane phospholipids (13). Similar observations have been made in the mammalian gastric epithelia where the canalicular surface of the parietal cell undergoes major alterations on stimulation of acid secretion. Relatively little is known, however, about the events responsible for the orderely transformation of these elements in the postulated (‘secretory apparatus” of the acid-secreting cell. Studies with a number of secretory tissues have implicated microtubules and microfilaments in the repetitive assembly, disassembly, and reassembly of the various structural elements involved in the secretion process. These cytoskeletal elements appear to be associated with several types of intracellular movements (3) including chromosome movement (9), transport and release of secretory products (15, 19, 20, 25, 27), and cytoplasmic streaming (18). The apical surface transformations in the E550
University
acid-secreting cell invoked by stimulation or inhibition of gastric H+ secretion appear to undergo somewhat similar movements. The antimitotic agents colchicine and vinblastine and others are known to exert disruptive effects on microtubules in vivo (9, 16) and to interact with microtubule subunit protein in vitro (2, 28). These agents have been employed extensively as tools in the study of microtubules in cellular functions. Cytochalasin B has been reported to disrupt microfilaments in several cell types (17) and has been shown to interact with actin (24). In an attempt to determine whether microtubules or microfilaments participate in the sequence of events leading to acid or pepsinogen secretion, we have studied the effects of these agents on H+ and pepsinogen secretions by the isolated frog gastric mucosal preparations. MATERIALS
AND
METHODS
Adult male frogs (Rana catesbeiam) were obtained from Mogul ED, Oshkoh, WI, and maintained unfed in running tap water for at least I wk before study. Colchitine was a product of Calbiochem, San Diego, CA; vinblastine and cytochalasin B were obtained from Sigma Chemical Co., St. Louis, MO. Hemoglobin (enzyme substrate powder) was supplied by Grand Island Biological Company, Grand Island, NY, and a 5% suspension of the powder was dialyzed extensively against 1.0 mM HCl at 4OC before use in pepsinogen assays. Burimamide was a generous gift from Dr. M. E. Parsons of Smith Kline & French Laboratories, London, England. All other chemicals were the highest grade purity available. Acid and pepsinogen secretion was monitored in isolated gastric epithelia mounted on polyethylene tubes. When electrical parameters were measured, chambered mucosal preparations were used. Briefly, the frog was pithed, the stomach removed and slit along its lesser curvature, and the mucosa separated from the smooth muscle coat by blunt dissection. The mucosa was divided longitudinally into approximately equal halves and one half was used as an appropriate control. Care was generally exercised to exclude esophageal and pyloric regions from the effective tissue area used in the study. The serosal and luminal bathing solutions were comprised of the frog Ringer solution (13) and 120 mM unbuffered NaCl, respectively, and both solutions were gassed with 95% 02-5s COZ. Acid secretion was measured with the pH stat method (4) at the end-point setting of 4.5 with 10 mM NaOH used for titration. Transepithelial poten-
0363-6100/79/0000-0000$01.25
Copyright 0 1979 the American Physiological Society
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COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E551
SECRETION
tial difference (PD) was monitored with a Kontron recorder with a pair of calomel half-cells connected to the chamber via balanced agar bridges. Resistance was calculated from the change in PD in response to passing a 2O+A current through a pair of saline agar bridges. Pepsinogen secretion was determined as pepsin activity in aliquots of luminal bathing media that were withdrawn completely every hour and replaced with fresh luminal solutions. Mucosal pepsinogen content was determined in aliquots of 10,000-g supernatant fractions of the homogenates prepared in 5 mM sodium acetate buffer, pH 4.0. Briefly, the pepsin assay procedure (I, 21) involved incubation of the enzyme with (final concentrations in 0.5 ml total volume) 2% hemoglobin in 0.1 N HCl as the substrate and 10 mM glycine buffer, pH 3.0 for 10 min at 37°C; the 0.1 N HCI used for diluting the hemoglobin buffered the incubation mixture finally at a pH of 1.8. Pepsin activity was terminated by the addition of 0.5 ml of 6% cold trichloroacetic acid (TCA). The hydrolysis products of hemoglobin contained in the filtrate as TCAsoluble tyrosine peptides were assayed by the method of Lowry et al. (14), with tyrosine used as standard. The enzyme activity was expressed as peptic units (PU), 1 PU representing an activity of pepsin that releases 0.1 pmol of tyrosine from 2% hemoglobin at pH 1.8 in 10 min at 37°C (7). The rate of secretion is expressed either as a) peptic units secreted per square centimeter per hour into the luminal bathing solution or b) the first-order rate constant for the decline of mucosal pepsinogen content. In the latter case, the rate constant was determined from the slope of the semilogarithmic plot of the pepsinogen remaining in the mucosa as a function of time. For this purpose, the mucosal pepsinogen content at zero time was calculated by adding the pepsinogen remaining in the mucosa at the end of experiment to the total amount secreted; subtracting the cumulative amounts of pepsinogen secreted at successive intervals yielded values for mucosal pepsinogen levels at the start of each interval. The expression of the pepsinogen secretion data as the first-order rate constant appear valid for the in vitro preparation, because little if any pepsinogen synthesis was observed under our experimental conditions in the absence of an amino acid supplement. This was established in preliminary experiments. Mucosae were incubated with 50 PCi of [3H]leucine (New England Nuclear, 60 Ci/mmol) in the serosal bathing medium for up to 6 h and the entire luminal solution (2-3 ml) containing secreted pepsinogen was loaded onto a Sephadex G-25 column. The column was eluted with 120 mM NaCl and 1.0~ml fractions were collected until pepsinogen appeared in the effluent, as determined by pepsin activity. No radioactive leucine was found in the fractions associated with pepsin activity, indicating that little de novo pepsinogen synthesis occurred under these experimental conditions. The relationship between pepsinogen and H+ secretion was expressed arbitrarily as a ratio, PU/H’. Resting gastric mucosal preparations were obtained as described previously (II). Various agents were added to the serosal bathing medium and, at concentrations of colchicine greater than 10 mM, isotonicity of the medium was adjusted bv a corresponding reduction in the
amounts of NaCl. All studies with colchicine out in the dark.
were carried
RESULTS
Effect of colchicine on H* secretion, PD, and resistance. Colchicine concentrations below 1 mM have no observable effects on acid or pepsinogen secretion in histamine-stimulated mucosae, At concentrations greater than I mM (IO-50 mM), there is a kinetically slow, biphasic effect on H+ secretion (Fig. 1). A transient initial stimulation of H+ secretion to a small but significant degree (l&30%) over that obtained with supramaximal histamine stimulus is followed by concentrationdependent inhibition. The inhibition can be reversed almost completely by repeated washouts of colchicine over a period of 2-3 h. In experiments in which the concentration of colchicine is increased stepwise from 0.01 to 50 mM at hourly intervals, each colchicine increment results in an initial rapid rise in PD followed by its return to base-line level over a period of 15-30 min (Fig. 2). The transmural resistance does not show significant changes until concentrations of colchicine above I mM are reached. The rise in resistance observed is consistent with the gradual decline of H+ secretion at higher colchicine concentrations. Prolonged incubation at higher colchicine concentrations eventually leads to a decline of short-circuit current, suggesting an inhibition of active chloride transport. Colchicine stimulation of H’ secretion in resting mucosa, Since the predominant effect of colchicine is to inhibit Hf secretion, it is difficult to explain the initial transient and significant stimulation. Furthermore, in most studies, as the inhibition of H+ secretion progressed at higher concentrations of colchicine, replacement of I
tSTAMlNE U4 COtCHIClNE
M
100
20 0
1
1
I
d501
4 6 8 HOURS FIG. 1. Effect of colchicine on H’ secretion, Mucosal halves from the same epithelium were stimulated with 1W4 M histamine 1 h after isolation. At the end of 2nd h, colchicine at indicated concentrations (1-50 mM) was added to one half while the second half was used as control. H+ secretion at the beginning of 3rd h was set arbitrarily to fUO% in each experiment and subsequent rates for both experimental and control halves were expressed as percentages of this rate. Insert shows dose dependence of inhibition 5 h after colchicine treatment. Vertical lines show t 1 SE for 3 or more determinations. 2
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D. K. KASBEKAR
E552 nutrient bathing medium with a fresh one containing colchicine resulted in a transient and partial reversal of inhibition. This transient stimulation of H+ secretion does not appear to be an artifact. In resting gastric mucosal preparations, colchicine has similar effects; it stimulates secretion from resting zero H+ secretion in the absence of any added secretagogues and this stimulation is followed by the usual pattern of inhibition seen in spontaneously secreting or histamine-stimulated mucosa
I
N E200 ;
‘e.*
100
-
lO-4 M HISTAMINE COLCHICINE hM)
0.010.1 I.0 p 20 A*’ ---a
l
-a-m-a
I
AND
G, S. GORDON
(Fig. 3). This stimulation may be due to the presence of a trace contaminant in colchicine, or colchicine per se may be a histamine releaser in the gastric mucosa and thus may stimulate secretion. Alternatively, colchicine may be metabolized by the mucosa and a metabolite may be responsible for the stimulatory effect. We have not pursued this aspect further and therefore have no explanation for the observed stimulation of H+ secretion by this agent. Colchicine effects on pepsinugen secretion and cumparison with H+ secretion. The fundic region of frog gastric mucosa contains and secretes pepsinogen in response to cholinergic stimuli, gastrin, and histamine (unpublished observations). The typical distribution of pepsinogen in the gastroesophageal region is shown in Fig. 4. An analysis of gastric and esophageal mucosae from five frogs yielded values of 3.4 t 0.3 and 12.1 f- 1.8 (mean t SE) PU/mg wet wt, respectively. Current evidence
4
I -
-I
‘-+-+,,,
l1 2
0
1 4
1 6
HOURS
1 0 FIG.
increasing Colchicine
2
4 I-OURS
6
3. Stimulation of resting H’ secretion by colchicine. Mucosal halves were brought to resting state by 18-h preincubation (not shown). Colchicine (10 mM) was added to one half (C) and 0.1 mM histamine (H) to the other at time indicated by arrow. In 2 other experiments colchicine-stimulated H+ secretion rates were similar and also declined faster with time compared with those obtained with corresponding histamine controls. FIG.
8
2. Typical response of PD, resistance, and H’ secretion to concentrations of colchicine in histamine-stimulated mucosa. concentrations were increased at hourly intervals as shown
1500
0 w
1000
F 3 u3
w 2 z-. 3
20 Distance
30 from
Pylorus
40
FIG. 4. Distribution of pepsinogen in gastroesophageal epithehuk Transverse strips (5 mm) were cut from the pyloric end, homogenized in acetate buffer, and assayed for pepsin activity.
50
(mm)
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COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E553
SECRET1ON
very strongly indicates that, unlike mammalian par&al cells, the frog tubular cell secretes both H+ and pepsinogen (12, 22). Since microtubules and microfilaments appear to be more intimately involved in the secretion of proteins in various tissues, we determined the effects of colchicine on pepsinogen secretion and compared the results with those on H+ secretion. The rates of H+ and pepsinogen secretion and the PU/H+ ratios for hourly intervals by the control and colchicine-treated mucosal halves are shown in Table 1 and Fig. 5, The data indicate that at 10 mM serosal colchicine concentration, pepsinogen secretion declines relatively more rapidly than H’ secretion, with a concomitant decline in PU/H+ compared with control without added colchicine. The PU/ H+ values for the control and colchicine-treated mucosal halves in four different studies were (mean t SE) 7.1 t I.0 and 1.5 & 0.3, respectively. This decline is mostly due to a decrease in the amount of pepsinogen secretion and not due to the initial small stimulation of H+ secretion observed with colchicine. These findings are in contrast to those noted with other inhibitors of Hf secretion, viz., thiocyanate or burimamide, where the decline in the rate of pepsinogen secretion lags significantly behind that of acid secretion (Fig. 6 and Table 2), resulting in increased PU/H+ ratios compared with the untreated controls. At
higher concentrations of colchicine, both H” and pepsinogen secretion rates decline rapidly, with little change in PU/H+. Effects of vinblastine on Hf andpepsinogen secretion. Vinblastine inhibition of pepsinogen and Hf secretion is shown in Fig. 7. Vinblastine (10w5-5 x 10m4M) is more effective than colchicine in inhibiting both secretions. Unlike colchicine, vinblastine does not stimulate H’ secretion transiently and the kinetics of inhibition of H+ secretion almost parallel that of pepsinogen, except at
mM 0
1 10 25 Values
are means
t4, h
k, h-’
0.124 0,117 0.092 0,046 0,035
k 1 SE for 4 or more
I
determinations.
10-4M
I.OmM
e
B’amide SCN-
~
7
5.7 * 0.2 5.9 -t- 0.4 7.5 t 0.3 15.0 -+ 0*2 20.0 -t- 0.2
50
0.1 mM Histomh
H ~20mM
TABLE 1. R&e constants for loss of mucosal pepsinogen in presence of colchicine in histamine-stimulated mucosae --.-- _ Colchicine,
I
FIG. 6. Effect of burimamide and thiocyanate on H’ rates in histamine-stimulated mucosae, Mucosal halves with 0.1 mM histamine and rates of H+ and pepsinogen observed for the next 4 h. Burimamide (1.0 mM) and mM) were then added to the individual halves and the studied for another 3 h. Note relatively slow decline secretory rates in contrast to H’ rates and compare colchicine in Fig. 5.
and pepsinogen were stimulated secretion were thiocyanate (20 secretory rates of pepsinogen with effects of
Histamine
owlthout
Colchlclnc
*with
Colchlclne
10
I
*
\
I
0 HrS-
I
I
4
8
FIG, 5. Effect of colchicine on histamine-stimulated H’ and pepsingen secretion. Mucosal halves were stimulated with histamine, and colchicine (10 mM) was added to one half while the other was used as control. Cross-hatched bars in top and bottom diagrams represent acid and pepsinogen secretion rates, refor the colchicine-treated spectively, half; open bars represent the corresponding controls. Insert shows PU/H+ for mucosal pair as a function of time. Note rapid decline of pepsinogen secretion in colchicine-treated half.
HOURS
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E554
D. K. KASBEKAR
AND
G. S. GORDON
2. PU/W ratios in histamine-stimulated mucasal halves beforeand after treatment with burimamide or tIz%ocyanate _._ _.._-------.~--.-..~--------- --- -,_._. __-.----.---- --.---- -------- W/H+ I TABLE
dependent inhibition of secretion with a concomitant rise in transmural resistance. The rate of decline of Hf secretion is also enhanced with increasing concentrations of the drug. In contrast, the decline in pepsinogen secretion is monophasic and precedes that of H’, as indicated by Before treatment After treatment decreasing PU/H’ ratios with time. Pepsinogen secretion 4h 5h 6h 7k 8h 9h is thus more sensitive to colchicine than acid secretion. Histamine, 0.1 4.9kO.8 6.3~1.0 6.ltl.I The effects of vinblastine, which also inhibits gastric mM secretion, differ in some respects from those of colchicine. + Burimamide, 36k-2 185k12 170-e-14 The concentrations required for complete inhibition of ImM both H* and pepsinogen secretion are considerably lower + Thiocyanate, 24-t-3 68-t- 7 207411 (5 x IF4 M) than those of colchicine, The initial tran20 mM --sient stimulation of H+ secretion observed with colchicine PU/H’ ratios are means t I SE for 4 experiments, similar to those is not seen with vinblastine. The decline of the H’ described in Fig. 6. Ifn each experiment, the 4th-, 5th-, and Gth-h periods served as control periods; at the end of the 6th h, one mucosal half was secretion rate with vinblastine almost parallels that of treated with burimamide and the other with thiocyanate. the pepsinogen secretion rate, so that the PU/H’ ratios do not show major changes as the inhibition of secretion \ progresses. On the other hand, the slow onset of inhibitoo tion is common to the two agents and the inhibition can be reversed in both cases by repeated washouts. The latter observation indicates that their inhibitory effect is not due to irreversible binding or damage to the tissue. Another interesting observation from the present study is the kinetics of colchicine inhibition of pepsinogen secretion vis-A-vis acid secretion in comparison with the effects of other classic inhibitors of acid secretion. Thus, thiocyanate and burimamide, which inhibit acid secretion, also inhibit pepsinogen secretion, With these agents, inhibition of pepsinogen secretion lags behind acid secretion so that there is an initial rapid increase in PU/H” as the inhibition progresses. On the other hand, as discussed above, with colchicine there is invariably a decrease in IO lC5 u4 c3 PU/H’, primarily as a result of greater decline in the VINBLASTINE (Ml rate of pepsinogen secretion. Since gastric secretogogues that stimulate H+ secretion also stimulate pepsinogen FIG. 7. inhibition of HW and pepsinogen secretory rates by vinblastine. Mucosal halves were stimulated by 10W4 M histamine, and one secretion in the frog gastric mucosa, colchicine dissociahalf was treated with vinblastine at indicated concentration while the tion of the two secretions from the same cell type may be other half was used as a control. Percent inhibition of secretion rates 5 useful in studying the point of divergence in the sequence h after vinblastine treatment was calculated as in Fig. 1. Numbers in of events leading to stimulation of the respective secreparentheses show average G.&-order rate constants (h-l) for pepsinotions by the gastric secretagogues. gen secretion. O-----O H+ secretion rate; t----o pepsinogen secretion rate. Vertical bars show I SE for 3 or more determinations. The morphological evidence for involvement of cytoskeletal elements in the gastric secretory process is, at present, merely correlative. Vial and Garrido (26) have higher concentration (5 x 1K4 M), when the rate of H’ secretion declines to zeru while pepsinogen leakage cun- demonstrated a change in the orientation of actin microtinues at a low basal rate. Like colchicine, however, the filaments with secretion and Forte and his colleagues (5) have shown the presence of microtubules in the mamkinetics of vinblastine inhibition are slow. Cytochalasin B (10-“-10S3 M) had no effect on either malian oxyntic cell just beneath the secretory surface in the apical region. They have also demonstrated cyclical pepsinogen or H+ secretion. changes in the microfilament orientation that parallel the changes in microvilli and tubulovesicular elements DISCUSSION with secretion. However, the microfilaments they deThe data presented above show that colchicine has scribe are not identified either as actin or the nonactin complex effects on gastric secretion. At concentrations filaments described in other tissues (6). A recent study greater than I mM, the drug has a biphasic action on by Hynes and Destree (8) indicates that nonactin filaacid but not on pepsinugen secretion. Within an hour of ments, although unaffected by cytochalasin B, are discolchicine addition, H” secretion increases. The stimu- rupted by colchicine. No report is made of the effects of lation, however, is transient and lasts for longer periods other agents such as vinblastine. at lower than at higher concentrations. Preliminary obThe data presented here can be considered as another servations indicate that in both the spontaneously se- line of correlative evidence supporting the contention creting and the resting mucosa this transient stimulation that cytoskeletal elements may be involved in the gastric by colchicine can be abolished by burimamide. The initial secretory response, whether they are microtubules or the increase in H’ secretion is followed by a concentrationmicrofilaments described by Hynes and Destree (8). ObDownloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 16, 2019.
COLCHICINE,
VINBLASTINE,
AND
GASTRIC
E555
SECRETION
viously binding studies must be performed in order to target the effects of these agents. This is also necessary for determining the basis of the relatively large concentrations of colchicine needed for secretory inhibition. The evidence at hand does not differentiate between permeability, metabolic destruction, or a large pool of unpolymerized subunits. However, both the morphological and physiological studies clearly indicate that this one facet
of the gastric secretory gation. The technical assistance,of edged. This study was supported
PCM 76 09971
process
warrants
Ms. Amita by National
Desai
further is gratefully
Science
Foundation
investiacknowlGrant
l
Received
24 August
1978; accepted
in final
form
21 December
1978.
REFERENCES
1. ANSON, M. L., AND A. E. MIRSKY. The estimation 2.
3. 4.
5.
6.
7.
8. 9.
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11, 12.
13.
14.
15,
of pepsin with hemoglobin. J. Gen. 1Dhysiol, 16: 59-63, 1932. BORISY, G. G., AND E. W. TAYLOR. The mechanism of action of colchicine: binding of colchicine-3H to cellular protein J, CeZZ BioL. 34: 525-533,1967. BUCKLEY, I. K., AND K. R. PORTER. Cytoplasmic fibrils in living cultured cells. Protoplasma 64: 349-380, 1967. DURBIN, R. P., AND E. HEINZ. Electromotive chloride transport and gastric acid secretion in the frog. J. Gen. PhysioZ. 41: 103% 1047,1958. FORTE, T. M., T. E. MACHEN, AND J. G. FORTE. Ultrastructural changes in oxyntic cells associated with secretory function. a membrane recycling hypothesis. GastroenteroZogy 73: 941-955, 1977. GOLDMAN, R. D., AND D. M. KNIPE. Function of cytoplasmic fibers in non-muscle cells. CoZd Spring Harbor Symp Quant. BioL 37: 523-534, 1972. HPRSCHOWITZ, B. I. Secretion of pepsinogen. In: Handbook of Physiology. Alimentary CanaL Washington, DC.: Am. Physiol. Sot. 1968, sect. 6, vol. II, p, 889-918. HYNES, R. O., AND A. T. DESTREE. Ten nm filments in normal and transformed celIs. Cell 13: 151-163, 1978. INOUI?, S., AND H. SATO. Cell motility by labile association of molecules: the nature of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 50, Suppl.: 259-292,1967. ITO, S., AND G. C. SCHOFIELD. Studies on the depletion and accumulation of microvihi and changes in the tubulovesicular compartment of mouse parietal. cells in relation to gastric acid secretion, J. CeZZ Biol, 63: 364-382, 1974. KASBEKAR, D. K. Studies of resting isolated frog gastric mucosa. Proc. Sot. Exp. Biol. Med. 125: 267-271, 1967, KASBEKAR, D. K., AND G. H. BLUMENTHAL. Isoltion, culture and some properties of frog gastric tubular cells. Gastroenterobgy 73: 881,1977. KASBEKAR, D. K., G. M. FORTE, AND J. G. FORTE. Phospholipid turnover and ultrastructural. changes in resting and secreting bullfrog gastric mucosa. Biochim. Biophys. Acta 163: l-13, 1968. LOWRY, 0. H., N. J. ROSEBROUGH, A, L. FARR, AND R, J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. &em. 193: 265-275,19X MALAISSE, W. J., F. MALAISSE-LAGAE, E. VAN OBBERGHEN, G. SOMERS, G. DAVIS, AND L. ORCI. The microtubular microffiamentous system of the pancreatic P-cell. Excerpta Med. Int, Congr,
Ser. 273: 282-287, 1973. 16. MALAWISTA, S. E., H. SATO, AND K, G. BENSCH. Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science 160: 770-772,1968. 17. MCGUIRE, J., AND G. MOELLMAN. Cytochalasin B: effects on microfilaments and movement of melanin granules within melanocytes. Science 175: 642-644, 1972. 18. NACHMIAS, V. T., H. E. HUXLEY, AND D. KESSLER. Electron microscope observations on actomyosin and actin preparations from Physarum polycephalum, and their interaction with heavy meromyosin subfragment I from muscle myosin. J. MoL BioZ. 50: 83-90, 1970. 19. ORR, T. S. C., D. E. HALL, AND A. C. ALLISON. Role of contractile microfilaments in the release of histamine from mast cells. Nature London 236: 350-351, 1972. 20. PAULSON, J. C., AND W. 0. MCCLURE. Inhibition of axoplasmic transport by colchicine, podophyllotoxin, and vinblastine: an effect on microtubules. Ann. NY Acad. Sci. 253: 517-527, 1975. 21. SCHLAMOWITZ, M., AND L. U. PETERSON. Studies on the optimum pH for the action of pepsin on “native” and denatured bovine serum albumin and bovine hemoglobin. J. BioZ. Chem. 234: 31373145, 1959. 22. SEDAR, A. W. Electron microscopy of the oxyntic cell in the gastric glands of the bullfrog (Rana catesbeiana). J. Biophys. Biochem. Cytol. 9: I-18, 1961. 23. SEDAR, A. W. Fine structure of the stimulated oxyntic cell. Federatiolz Proc. 24: 1360-1367, 1965. 24. SPUDIK, J. A., AND S. LIN. Cytochalasin B, its interaction with actin and actomyosin from muscle. Proc. NatZ. Acad. Sci. USA 69: 442-446, 1972. 25. THOA, N. B., G. F. WOOTEN, J. AXELROD, AND I. J. KOPIN. Inhibition of release of dopamine P-hydroxylase and norepinephrine from sympathetic nerves by colchicine, vinblastine or cytochalasin 13. Proc. NatL Acad. Sci. USA 69: 520-522, 1972. 26. VIAL, J. D., AND J. GARRIDO. Actin-like filaments and membrane rearrangement in oxyntic cells. Proc. NatZ. Acad. Sci. USA 73: 4032-4076, 1976. 27. WILLIAMS, J. A., AND J, WOLFF. Possible role of microtubules in thyroid secretion. Proc. NatZ. Acad. Sci. USA 67: 1901-1908, 1970. 28. WILSON, L. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyhotoxin. Biochemistry 9: 4999-5007, 1970.
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