Aspirin induces morphological transformation to the secretory state in isolated rabbit parietal cells

GASTROENTEROLOGY

1991;101:404-409

Aspirin Induces Morphological Transformation to the Secretory State in Isolated Rabbit Parietal Cells UMA

K. MURTHY

and ROBERT

A. LEVINE

Section of Gastroenterology, Department of Medicine, State University of New York Health Science Center and Veterans Administration Medical Center, Syracuse, New York

The morphological response of rabbit parietal cells to aspirin was evaluated by grading several ultrastructural features including the extent of the tubulovesicular system, intracellular secretory canaliculi, and microvilli. After exposure of isolated parietal cells and gastric glands to aspirin or histamine, there was an approximately twofold increase in the ratio of secretory to nonsecretory parietal cells, and depletion of extracellular Ca2+ abolished the aspirin-induced morphological changes. Morphometry in parietal cells showed that aspirin induced a sixfold increase in secretory canalicular membrane elaboration. Aspirin potentiated histamine-induced parietal cell respiration and aminopyrine uptake ratio but did not increase basal respiration or aminopyrine uptake, suggesting an apparent dissociation from aspirin-induced morphological changes.

ehave

previously observed that aspirin potentiates secretagogue-stimulated acid secretion in isolated rabbit fundic glands (FGs) and parietal cells (PCs] without affecting basal acid secretion (1).Our studies indicated that aspirin acts at a postreceptor site, beyond adenylate cyclase but before the proton pump. The initial potentiating effect of aspirin was dependent on intracellular calcium (Ca”), but its subsequent maintenance of secretion was dependent on extracellular Ca’+. During secretagogue-induced acid secretion, PC, undergo striking morphological changes (2-5).The present study was undertaken to evaluate whether aspirin treatment induced similar changes in PCs that might correlate with its augmentation of prestimulated acid secretion. Our results show that aspirin induces morphological changes indicative of the secretory state by a Ca’+-dependent process.

W

Materials and Methods Isolation and Viability of Fundic Glands and Parietal Cells Fundic glands and PCs were isolated from fasting New Zealand white rabbits (2.5-3.0 kg] using the method of Berglindh and Obrink (6) for FGs and a modification (7) of Berglindh’s method (8) for PCs. Viability of FG or PC suspensions was determined by erythrosin B dye exclusion, and only those having > 90% viability were used. Functional viability of PCs was assessed in most experiments using aminopyrine (AP) uptake ratio (7) and/or PC respiration (S), as previously described. The latter is an alternative functional equivalent to measurement of AP uptake. Histamineand aspirin-stimulated

oxygen consumption production (9).

was indirectly

measured

as 14C0,

Drug Treatment of Fundic Glands and Parietal Cells In the first set of experiments, isolated PCs and FGs were washed three times in an oxygenated, Ca’+-containing medium, and 1-mL aliquots containing a suspension of FGs or PCs (106) from the same animal were incubated for 20 minutes at 37°C with aspirin (10 p,mol/L), histamine (100 pmol/L), salicylic acid (10 kmol/L), or respiratory medium (control). In the second set of experiments, to deplete three times in a extracellular Ca’+, PCs were washed Ca’+-free respiratory medium containing 0.2 mmol/L EGTA before a final wash in a Ca’+-free medium. Parietal cells were then resuspended in Ca’+-supplemented or Ca’+depleted respiratory medium alone for 5 minutes at 37°C. After the 5-minute preincubation, l-mL aliquots of PC suspensions were incubated for 6 minutes in the absence or

Abbreviations used in this paper: AP, aminopyrine;

dibutyryl cyclic adenosine

monophosphate;

dbcAMP, FG, fundic gland: PC,

parietal cell; SC, secretory canaliculi; TV, tubulovesicle. o 1991 by the American Gastroenterological Association 0016-5065/91/$3.00

August 1991

ASPIRIN-INDUCED MORPHOLOGICALCHANGES IN PARIETAL CELLS 405

presence of histamine (100 Fmol/L), dibutyryl cyclic adenosine monophosphate (dbcAMP) (I mmol/L), and aspirin (10 pmol/L), washed with buffer, centrifuged, and prepared for electron microscopy. In a third set of experiments, isolated PCs were preincubated with 10 kmol/L cimetidine for 10 minutes at 37°C and then washed in respiratory buffer before a ZO-minute exposure to histamine (100 kmol/L) and/or aspirin (10 kmol/L).

Ultrastructural Studies Fundic glands or PCs were fixed either 6 or 20 minutes after initiation of drug treatment in a solution of 2% formaldehyde and 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide at 4”C, dehydrated in a series of graded ethanol, and embedded in epoxy resin. Thin sections were cut on a Sorvall MTXB ultramicrotome (DuPont, Newtown, CT), mounted on copper grids, stained with uranyl acetate and lead citrate, and examined under a JEOL-100SX (Peabody, MA) electron microscope. Sections were initially examined to ensure that the preparations contained 80%~90% PCs. Ultrastructural features of 50-100 consecutive PCs were blindly evaluated in each experiment, and PCs were classified as nonsecretory, intermediate, or secretory. Parietal cells with an abundance of smooth-surfaced tubulovesicles (TVs) in the cytoplasm and only a few collapsed intracellular canaliculi were considered nonsecretory. Secretory PCs were cells with an extensive system of intracellular (secretory) canaliculi (SC) lined by numerous long, slender microvilli with a concomitant reduction in the TV system (Figure 2A). Intermediate or partly stimulated PCs were those with features of both nonsecretory and secretory cells, i.e., a well-developed SC system and the persistence of TVs (4). For morphometric analysis, one thin section from each treatment group was chosen at random to be photographed. The section was examined systematically, starting at one corner, and the first lo-20 nucleated, well-preserved PCs found were photographed at the magnification that included the entire cross section of the cell (4000-5000~). The electron micrographs were enlarged to a final magnification of between 12,000 and 18,000X. Profiles of the entire cell, its nucleus, mitochondria, SC, and TV were outlined and digitized on an image analyzer (ZIDAS LEITZ, New York, NY) that computed the area in square millimeters. Only cells with nuclei occupying at least 3% of the area of the cell section were analyzed (3). Cells with vacuolated mitochondria and swelling or disruption of cytoplasmic membranes were also excluded (3,lO). Digitization was duplicated on every fifth PC to calculate the method error (11).

Materials Sodium salicylate, aspirin, and histamine dihydrochloride were obtained from Sigma Co. (St. Louis, MO). Aspirin and salicylic acid were dissolved in 50-100 PL of IO mmol/L sodium hydroxide and adjusted to pH 7.5 using I mmol/L hydrochloric acid after dilution in water. Dibutyryl

CAMP was obtained from Boehringer Mannheim (Mannheim, Germany). Cimetidine was purchased from Smith Kline Laboratories (Philadelphia, PA). All chemicals were of the highest grade available.

Statistical Evaluation The number of secretory PCs in each treatment group was expressed as a percentage of the total PCs counted (mean * SEM). Results from data analysis counting secretory and intermediate PCs together did not differ from that using secretory PCs alone. Therefore, intermediate PCs were included with secretory PCs. For morphometric analysis in each cell, the area occupied by SC was expressed as a percentage of that occupied by SC and TV, the two components of primary interest in this study [SC (mm’)/SC + TV (mm”) x 1001. The means ~frSEM were calculated for all the cells in each of the treatment groups. Analysis of variance and multiple comparisons between treatment groups were performed using Student’s NewmanKeuls test, using a software package with a limit of significance set at < 0.05.

Results Aspirin (10 Fmol/L) did not affect basal respiration, but it potentiated maximal histamine (100 pmol/ L)-stimulated respiration (‘CO, production) by 50% (P < 0.05, n = 7) at 30 minutes. The values were calculated by measuring stimulated minus basal “C0, production and set at 100%. Basal values with and without aspirin and histamine with and without aspirin were, respectively, 34 + 2 and 34 ? 1 and 55 2 2 and 48 ? 1 nmol/mg dry wtih. Similarly, basal AP uptake was unaffected, but histamine-stimulated AP uptake was potentiated 106% ‘-c 19% by aspirin (P < 0.05, n = 3). Aspirin after 20 minutes of treatment, like histamine, induced morphological transformation of resting PCs and FGs to the secretory state (Figures 1 and 2A). The 2.5- and z-fold increases, respectively, in secretory PCs and FGs induced by aspirin and histamine were significantly different from those in control and salicylic acid treatments (P < 0.05). Morphometric analysis of 138 cells from four animals (Figure 3) showed that aspirin, histamine, and aspirin plus histamine elicited a sixfold increase in the elaboration of SC membranes (P < 0.05). The method errors were 7.9 x lo-’ and 6.0 x 10-fi SD, respectively, for SC and TV. The effects of depletion of extracellular Ca2+ are shown in Figure 4. Parietal cell incubation with aspirin in Ca2+ -supplemented buffer for 6 minutes increased the percentage of secretory PCs, as did incubation with histamine and dbcAMP (P < 0.05). In the absence of extracellular Ca”, the secretory response to aspirin was abolished (P < 0.05). How-

406 MURTHY AND LEVINE

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Fundic Glands

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Control

SA

ASA

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Hist

Control

SA

ASA

Hist

Figure 1. Morphological response of isolated rabbit PCs and FGs to salicylic acid (SA, 10 pmom), aspirin (ASA, 10 pmoVL), and histamine (Hist, 100 bmol/L) compared with resting controls. *P < 0.05. n = experiments in each group. Values in this and subsequent figures expressed as mean f SEM.

at 20 minutes (1) appear to be temporally related. Although we believe that Ca2+ flux contributes to the aspirin effect on morphology and enhancement of secretagogue-stimulated acid secretion, the interrelationships are not necessarily directly proportional. For example, in isolated rabbit PCs, whereas carbachol and histamine produce similar changes in ultrastructure (unpublished observations) compared with carbachol, histamine induced a 25% less increase in intracellular free Ca’+ but a 75% greater stimulation of acid secretion (1). A similar Ca’+-modulating role of aspirin on PC morphology may be proposed, although the exact mode of action remains to be elucidated. The morphological changes induced by histamine have also been reported to show some Ca*+ dependency (12) and possible linkage to CAMP (13,14). Whereas aspirin induces secretory ultrastructural changes, we have been unable to functionally show the augmentation of basal PC respiration or AP uptake by aspirin in this and previous studies, although Discussion aspirin did enhance prestimulated-acid secretion. We believe that this is an apparent rather than a true This study shows that aspirin in isolated rabbit dissociation between morphology and function in PCs and FGs elicits the morphological transformation PCs. Some degree of spontaneous stimulation appears associated with stimulation of PC function. We showed inherent in our PC preparation, as supported by the a statistically significant difference between resting fact that 20% of the PCs in untreated preparations PC morphology and aspirin- or secretagogue-induced were secretory. Because our preparations were only ultrastructural changes, as evaluated by the increase 85% PC pure, they could be subject to residual in the percentage of secretory PCs and an increase in mast-cell stimulation. Thus, morphological effects of the elaboration of SC membrane by morphometry. aspirin could result from a synergistic effect with The specificity of the effect of aspirin has been shown histamine present in the preparation but inadequate using salicylic acid, which did not change resting PC to produce a demonstrable effect on function as morphology. Furthermore, the morphological changes measured by PC respiration and AP uptake. Further induced by aspirin were shown to be calcium depenexplanations for the apparent dissociation could be dent. We had previously shown that aspirin mobilized intracellular free Ca’+ within seconds in PCs (1). the effect of aspirin on Ca’+, a reduction in prostanoid synthesis, and/or the effects of the cell-dispersion Minute changes in CaZf can induce functional and ultrastructural changes in PCs. The aspirin-induced technique. As with aspirin, under other circumstances, it has been shown that although morphologiCa2+ flux, which occurs within seconds (l),the cal changes are a prerequisite for acid secretion, they ultrastructural transformation, which occurs at 6 precede (2,4,15), can occur independently of (10,13), minutes, and the functional changes that are maximal

ever, histamine- and dbcAMP-stimulated responses were not significantly altered. Depletion of CaZf was not associated with obvious ultrastructural changes in mitochondrial or other cytoplasmic organelles (Figure 2B). Parietal cells in the basal state and in response to aspirin, histamine, and dbcAMP showed wide variations in morphology, including differences in shape and number of surface microvilli and vacuoles. The spectrum of secretory changes ranged from maximally secretory (Figure 2A) to PCs with less developed SC systems and persistence of some TV; SC were occluded with microvilli or were large, dilated, and lined by few microvilli. Pretreatment of PCs with cimetidine did not decrease the number of secretory PCs in the basal state pretreatment = 14%; (control = 16%; cimetidine n = 2).

August

ASPIRIN-INDUCED

1991

Figure 2. A. Electron micrographs expanded intracellular SC system, x8500); M, mitochondria. B. Nonsecretory

PC [aspirin

MORPHOLOGICAL

of secretory PCs [aspirin (10 pmol/‘L)-treated, densely packed with numerous microvilli

(10 pmol/L)-treated,

Ca”-depleted]

showing

CHANGES

IN PARIETAL

CELLS

407

Ca’+ -supplemented] showing virtual absence of TV and an (mv) surrounding the nucleus (N) (original magnification

predominantly

an extensive

TV system

(original

magnification

X6800).

and are not necessarily directly proportional to (12,13) acid secretion. We and others (15,161 believe that functional tests and ultrastructural appearance should be used in conjunction to better evaluate the secretory response in PCs. Functional assessment of PC secretion by AP uptake ratio is only indirect and may not be sufficiently sensitive to detect small changes in acid accumulation in the resting isolated PC with a low pH This may be gradient (17)and loss of cell polarity (16). the case in aspirin-treated PCs in which we have observed evidence of acid secretion by morphological transformation but not by AP uptake. Even after 5-6 minutes in the presence of a secretagogue such as histamine, at which time morphological changes are evident, AP uptake in PCs is unchanged and only increases significantly after 15-20 minutes. In other instances, augmented AP uptake or respiration in PCs may be unaccompanied by ultrastructural changes and may not truly indicate acid secretion (15,16). The wide range in rabbit PC morphology observed in basal and secretagogue-stimulated preparations appears to reflect that of PCs in various stages of

secretion, similar to those described by Carlisle et al. in frog gastric mucosal preparations (13) and by Ito et al. in isolated mouse PCs (16). A possible explanation for this variability may relate to the isolation procedure per se, which disrupts tight junctions (16).In addition, secretagogues including histamine may not uniformly produce extensive changes in cell structure 151. Our study probably underestimates the degree of secretory changes because of our inability to inhibit basal secretory tone with cimetidine, to reduce the percentage of secretory cells, and to start experiments with a uniformly resting PC population. This may again reflect the inherent secretory ability of this PC preparation and the fact that cimetidine was only used as a pretreatment and washed off before histamine and aspirin exposure. When used in the same doses but left in the preparation during treatment with histamine and aspirin, acid secretion was inhibited (1). A similar inability with an Hz-receptor antagonist to inhibit secretory tone, as measured by basal oxygen uptake, has been shown by Sol1 in isolated canine PC (18).

408 MURTHY

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GASTROENTEROLOGY

AS A

Hist + ASA

Hist

Figure 3. Morphometric analysis of SC membrane elaboration in response to aspirin (ASA, 10 pmoliL), histamine (Hist, 100 pmol/ L), and histamine plus aspirin (Hist & ASA) compared with resting controls (P < 0.05); n = no. of cells from four animal preparations in the aspirin-treated, control, and histamine-treated groups and from three animals in the histamine + aspirin-treated group.

These morphological observations and our prior demonstration that aspirin augmented prestimulatedacid secretion in PCs (1)suggest that aspirin acts as a potential secretagogue modulator in vitro. Although nonsalicylate nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to increase gastric acid secretion in vitro and in vivo (19-M), the role of aspirin in vivo is much more complex than in PCs because of its conversion by mucosal esterases to salicylic acid. In vivo studies have reported an inhibition of gastric acid secretion by both aspirin and salicylic acid (25-27). We have found in the chambered frog gastric mucosa (28), dependent on concentration, either augmentation or inhibition of histaminestimulated acid secretion with 10 pmol/L and 10 mmol/L aspirin, respectively. This could relate to the variable conversion to salicylic acid with the different

H

+Ca*+ -Ca*+

dbcAMP

ASA

0

Control

Hi.9

Figure 4. Morphological response of isolated rabbit PCs to histamine (Hist, 100 urnof&, dbcAMP (1 mmol/L), and aspirin [ASA, IO umol/L) compared with resting controls in the presence and absence of Ca’+, *P < 0.05. tP < 0.05 for aspirin + Ca’+ vs. aspirin - Caz+; n = 3 experiments in each group.

Vol. 101, No. 2

concentrations of aspirin. In this study, we found no effect of 10 kmol/L salicyclic acid on isolated rabbit PCs. In frog mucosa, 5 mmol/L salicylic acid inhibited histamine-stimulated secretion, confirming studies by others (25) and showing that salicylic acid concentrations > 2.5 mmol/L were injurious to frog mucosa (25). Thus, it is possible that aspirin in lower concentrations may be directly responsible for injury because of the enhancement of acid secretion or the alteration of prostaglandin synthesis. At higher concentrations, aspirin injury may relate to its conversion to salicylic acid. In vivo actions are further affected by modulation of mucosal blood flow by both salicylate and nonsalicylate NSAIDs (29) and by the interaction of acidstimulating and acid-inhibiting paracrine, vascular, and neural influences. Although enhancement by aspirin of basal acid secretion (30) and meal-stimulated gastric PC secretion has been shown in healthy volunteers (31), it remains to be established whether or not aspirin modulates stimulus-secretion coupling processes in humans.

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25. Rowe PH, Starlinger MJ, Kasdon E, Marrone G, Silen W. Effect of simulated systemic administration of aspirin, salicylate and indomethacin on amphibian gastric mucosa. Gastroenterology 1986;90:559-569. 26. Rowe PH, Starlinger MJ, Kasdon E, Hollands MJ, Silen W. Parenteral aspirin and sodium salicylate are equally injurious to the rat gastric mucosa. Gastroenterology 1987;93:863-871. 27. Shea-Donohue T, Steel L, Montcalm-Mazzilli E, Dubois A. Aspirin-induced changes in gastric function: role of endogenous prostaglandins and mucosal damage. Gastroenterology 1990;98:284-292. 28. Nandi J, Levine RA. Aspirin (ASA) augments prestimulated acid secretion in chambered frog gastric mucosa (abstr). Gastroenterology 1990;98:A95. 29. Kauffman GL, Aures DA, Grossman MI. Intravenous indomethatin and aspirin reduce basal gastric mucosal blood flow in dogs. Am J Physiol1980;238:G131-G134. 30. Hunt JN, Smith JL, Jiang CL, Kessler L. Effect of synthetic prostaglandin E, analog on aspirin-induced gastric bleeding and secretion. Dig Dis Sci 1983;28:897-902. 31. Myers BM, Smith JL, Graham DY. Effect of red pepper and black pepper on the stomach. Am J Gastroenterol1987;82:211214.

Received March 15,199O. Accepted December 19,199O. Address requests for reprints to: Uma K. Murthy, M.D., Department of Medicine, University Hospital, 750 East Adams Street, Syracuse, New York 13210. This study was presented in part at the 1987 Annual Meeting of the American Gastroenterological Association and appeared in abstract form (Gastroenterology 1987;92:1547). The authors thank Dr. Jyotirmoy Nandi, Dr. Douglas Robertson, and Rebecca L. King for expert advice and laboratory assistance.