Role of mucus in gastric mucosal protection.

Even though there is no general agreement as to the mechanism of gastric mucosal protection, the consensus is that the initial brunt of luminal insults falls on the mucus layer which constitutes the only identifiable physical barrier between the gastric lumen and the mucosal surface. The continuous renewal and resilient nature of this layer efficiently counters peptic erosion of the gel, assures its viscoelastic and permselective properties, and provides a milieu for containment of the diffusing luminal acid by mucosal bicarbonate. Disturbances in this delicate balance lead to the impairment of the protective function of mucus resulting in gastric disease. Indeed, the weakening of gastric mucosal defense is intimately associated with the diminished viscoelastic qualities of mucus, decrease in hydrogen ion retardation capacity, and the extensive proteolysis of its mucin component. Although until recently the disintegration of the mucus coat was attributed exclusively to the enhanced activity of intragastric pepsin, our studies provided strong argument that a bacterial factor, namely infection by Helicobacter pylori, through the action of its protease and lipase enzymes also is highly detrimental to the integrity of gastric mucus. Hence, agents capable of interfering with the pathogenic activity of this bacteria are becoming the drugs of choice in peptic ulcer therapy.     

乙醇对胃粘膜的损伤及牛磺酸的保护作用

为了探讨乙醇性胃粘膜损伤和牛磺酸对其保护作用的机制,将４０只ＳＤ大鼠分为对照组、乙醇损伤组和牛磺酸保护组,观察其胃粘膜中内皮素（ＥＴ）、一氧化氮合成酶（ＮＯＳ）、生长抑素（ＳＳ）和血管活性肠肽（ＶＩＰ）的变化。结果如下：（１）随着乙醇作用时间延长、粘膜的损伤加重,胃粘膜内ＥＴ含量显著上升,而ＮＯＳ、ＳＳ和ＶＩＰ含量显著下降;（２）牛磺酸可显著减轻乙醇性胃粘膜损伤,抑制ＥＴ释放,增加ＮＯＳ活性和ＳＳ、ＶＩＰ的含量。上述结果表示,ＥＴ、ＮＯＳ、ＳＳ和ＶＩＰ的变化参与了乙醇性胃粘膜损伤的病理生理过程;牛磺酸对乙醇性胃粘膜损伤具有保护作用,可能与其调节胃粘膜内ＥＴ、ＮＯＳ、ＳＳ和ＶＩＰ的释放与活性有关。     

Role of sulfhydryls and early vascular lesions in gastric mucosal injury

This paper reviews the recently discovered role of sulfhydryls and early vascular injury in the pathogenesis of acute gastric mucosal injury. In the rat ethanol caused a dose-dependent decrease in nonprotein sulfhydryl concentration in the gastric mucosa within 1-5 min following an intragastric dose. These biochemical changes were accompanied by increased vascular permeability in the glandular stomach as revealed by the measurement of extravasated Evans blue injected i.v. prior to the administration of ethanol. Morphologic evidence of vascular injury was provided by labelling of damaged blood vessels in the stomach following the i.v. administration of colloidal particles in the form of india ink or monastral blue. The functional and structural damage to capillaries and venules in the glandular stomach was also maximal within 1-6 min after 1 ml of 75 or 100% ethanol given orally. Pretreatment with sulfhydryl (SH) containing drugs (e.g., L-cysteine, N-acetyl-L-cysteine, cysteamine, dimercaprol) or prostaglandin (PG) F2 beta prevented the ethanol-induced increase in vascular permeability, the labelling of blood vessels with vascular tracers, and the subsequent haemorrhagic erosions. The desquamation of superficial epithelial cells, however, was not markedly modified by either SH or PG compounds. This organoprotective effect of SH and PG drugs was virtually counteracted in adrenalectomized rats that exhibited "vascular fragility". Glucocorticoid treatment restored the response of adrenalectomized animals. Thus, a SH- and glucocorticoid-sensitive early vascular injury seems to be of major significance in the pathogenesis of haemorrhagic gastric erosions and SH-containing compounds represent a new group of cytoprotective or organoprotective agents.     

Role of endogenous nitric oxide synthase inhibitor in gastric mucosal injury

To explore the role of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in gastric mucosal injury, 3 models of gastric mucosal injury induced by ethanol, indomethacin, or cold stress were used in rats. The cultured human gastric mucosal epithelial cell line GES-1 infected by Helicobacter pylori (Hp) was selected to mimic human gastric mucosal injury. Gastric mucosal ulcer index (UI), levels of ADMA and NO, and activity of dimethylarginine dimethylaminohydrolase (DDAH) were determined in the mucosal injury models; in Hp-infected or ADMA-treated GES-1 cells, levels of ADMA, NO, and TNF-alpha and activity of DDAH were measured. The results showed that UI and levels of ADMA were markedly increased and accompanied by significantly decreased DDAH activity in the mucosal injury models. Incubation of GES-1 cells with Hp increased levels of TNF-alpha and ADMA and decreased activity of DDAH. Administration of ADMA also increased levels of TNF-alpha. The results suggest that ADMA plays an important role in facilitating gastric mucosal injury, an effect which is associated with inhibiting NO synthesis and inducing inflammatory reaction.     

Role of mucus in ischemia/reperfusion-induced gastric mucosal injury in rats

Gastric mucus plays an important role in gastric mucosal protection. Apart from its "barrier" function, it has been demonstrated that mucus protects gastric epithelial cells against toxic oxygen metabolites derived from the xanthine/ xanthine oxidase system. In this study, we investigated the effect of malotilate and sucralfate (mucus production stimulators) and N-acetylcysteine (mucolytic agent) on ischemia/reperfusion-induced gastric mucosal injury. Gastric ischemia was induced by 30 min clamping of the coeliac artery followed by 30 min of reperfusion. The mucus content was determined by the Alcian blue method. Sucralfate (100 mg/kg), malotilate (100 mg/kg), and N-acetylcysteine (100 mg/kg) were given orally 30 min before surgery. Both sucralfate and malotilate increased the mucus production in control rats. On the other hand, N-acetyloysteine significantly decreased mucus content in control (sham) group. A significant decrease of mucus content was found in the control and the N-acetylcysteine pretreated group during the period of ischemia. On the other hand, sucralfate and malotilate prevented the decrease the content of mucus during ischemia. A similar result can be seen after ischemia/reperfusion. In the control group and N-acetylcysteine pretreated group a significant decrease of adherent mucus content was found. However, sucralfate and malotilate increased mucus production (sucralfate significantly). Sucralfate and malotilate also significantly protected the gastric mucosa against ischemia/reperfusion-induced injury. However, N-acetylcysteine significantly increased gastric mucosal injury after ischemia/reperfusion. These results suggest that gastric mucus may be involved in the protection of gastric mucosa after ischemia/reperfusion.     

Presenilins: Role in calcium homeostasis

Mutations in presenilins are responsible for the vast majority of early-onset familial Alzheimer's disease cases. Full-length presenilin structure is composed of nine transmembrane domains which are localized on the endoplasmic reticulum membrane. Upon endoproteolytic cleavage, presenilins assemble into the γ-secretase multiprotein complex and subsequently get transported to the cell surface. There is a wealth of knowledge around the role of presenilins as the catalytic component of γ-secretase, their involvement in amyloid precursor protein processing and generation of neurotoxic β-amyloid species. However recent findings have revealed a wide range of γ-secretase-independent presenilin functions, including involvement in calcium homeostasis. Particularly, familial Alzheimer's disease presenilin mutations have been shown to interfere with the function of several molecular elements involved in endoplasmic reticulum calcium homeostasis. Presenilins modulate the activity of IP(3) and Ryanodine receptor channels, regulate SERCA pump function, affect capacitative calcium entry and function per se as endoplasmic reticulum calcium leak conductance pores.     

Role of cyclooxygenase isoforms in gastric mucosal defence.

A complex system of interacting mediators exists in the gastric mucosa to strengthen its resistance against injury. In this system prostaglandins play an important role. Prostaglandin biosynthesis is catalysed by the enzyme cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. Initially the concept was developed that COX-1 functions as housekeeping enzyme, whereas COX-2 yields prostaglandins involved in pathophysiological reactions such as inflammation. In the gastrointestinal tract, the maintenance of mucosal integrity was attributed exclusively to COX-1 without a contribution of COX-2 and ulcerogenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) were believed to be the consequence of inhibition of COX-1. Recent findings, however, indicate that both COX-1 and COX-2 either alone or in concert contribute to gastric mucosal defence. Thus, in normal rat gastric mucosa specific inhibition of COX-1 does not elicit mucosal lesions despite near-maximal suppression of gastric prostaglandin formation. When a selective COX-2 inhibitor which is not ulcerogenic when given alone is added to the COX-1 inhibitor, severe gastric damage develops. In contrast to normal gastric mucosa which requires simultaneous inhibition of COX-1 and COX-2 for breakdown of mucosal resistance, in the acid-challenged rat stomach inhibition of COX-1 alone results in dose-dependent injury which is further increased by additional inhibition of COX-2 enzyme activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. COX-2 inhibitors do not damage the normal or acid-challenged gastric mucosa when given alone. However, when nitric oxide formation is suppressed or afferent nerves are defunctionalized, specific inhibition of COX-2 induces severe gastric damage. Ischemia-reperfusion of the gastric artery is associated with up-regulation of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone augment ischemia-reperfusion-induced gastric damage up to four-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE(2). Selective inhibition of COX-1 is less effective. Furthermore, COX-2 inhibitors antagonize the protective effect of a mild irritant or intragastric peptone perfusion in the rat stomach, whereas the protection induced by chronic administration of endotoxin is mediated by COX-1. Finally, an important function of COX-2 is the acceleration of ulcer healing. COX-2 is up-regulated in chronic gastric ulcers and inhibitors of COX-2 impair the healing of ulcers to the same extent as non-selective NSAIDs. Taken together, these observations show that both COX isoenzymes are essential factors in mucosal defence with specific contributions in various physiological and pathophysiological situations.     

Role of cyclooxygenase-2 in gastric mucosal defense.

Two isoenzymes of cyclooxygenase (COX), the key enzyme in prostaglandin (PG) biosynthesis, COX-1 and COX-2, have been identified. COX-1 was proposed to regulate physiological functions, COX-2 to mediate pathophysiological reactions such as inflammation. In particular, it was suggested that maintenance of gastric mucosal integrity relies exclusively on COX-1. Recently, it was shown that a selective COX-1 inhibitor does not damage the mucosa in the healthy rat stomach, although mucosal prostaglandin formation is near-maximally suppressed. However, concurrent treatment with a COX-1 and a COX-2 inhibitor induces severe gastric damage. This indicates that in normal mucosa both COX-1 and COX-2 have to be inhibited to evoke ulcerogenic effects. In the acid-challenged rat stomach inhibition of COX-1 alone is associated with dose-dependent injury which is aggravated by additional inhibition of COX-2 activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. After acid exposure, COX-2 inhibitors cause substantial gastric injury when nitric oxide formation is suppressed or afferent nerves are defunctionalized. Ischemia-reperfusion of the gastric artery increases levels of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone aggravate ischemia-reperfusion-induced mucosal damage up to 4-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE2. Furthermore, the protective effects elicited by a mild irritant or intragastric peptone perfusion are antagonized by COX-2 inhibitors. Finally, COX-2 expression is increased in experimental ulcers. COX-2 inhibitors delay the healing of chronic gastric ulcers in experimental animals and decrease epithelial cell proliferation, angiogenesis and maturation of the granulation tissue to the same extent as non-steroidal anti-inflammatory drugs. These observations indicate that, in contrast to the initial concept, COX-2 plays an important role in gastric mucosal defense.     

Impact of ethanol on innate protection of gastric mucosal epithelial surfaces and the risk of injury.

Earlier investigations on the effect of ethanol on synthesis and posttranlational glycosylation of gastric mucus glycoprotein (mucin) revealed quantitative changes in the apoprotein assembly, glycosylation, and mucin retention on the mucosal surface (Slomiany et al.., Alcoholism: Clin. Exp. Res. 21, 417-423, 1998). To assess whether metabolic consequences of ethanol ingestion, documented in the in vitro system are also occurring in vivo the rats were subjected to 8 weeks of ethanol containing liquid diet. The retention of mucin on the surface of gastric mucosa was quantitated by measuring the binding of gastric mucin to Mucin Binding Protein (MBP) of gastric mucosa. The results were compared with those obtained with the rats subjected to pair-feeding the isocaloric-control diet. Before alcohol administration, and in two weeks' intervals thereafter, the gastric contents from the animals was collected and mucin purified. After 8 weeks of the respective diet, the animals were sacrificed and their gastric mucosa used for MBP preparation. The binding of mucin to MBP before ethanol, and after 2, 4, 6, and 8 weeks of ethanol diet was quantitated with Enzyme Linked Lectin Assay (ELLA). The study with standard mucin revealed that binding of mucin to MBP differs substantially between individual animals. The same variability in binding was observed with the individual mucin preparations collected at the onset of the experiment. However, with the progression of ethanol feeding, the mucin samples besides displaying the variable and animal-specific binding to MBP at the initiation of the experiment, also showed a dramatic decrease in binding. In five animals, after two weeks of ethanol diet, mucin binding to MBP decreased by 50%; in two animals, the drastic decrease in binding was observed in mucin collected after four weeks of alcohol feeding; and in one animal a 20% decrease in binding persisted for six weeks, and then decreased to 50% in the last collection. Also, in two animals, the mucin collected after 8 weeks of ethanol feeding retained only 6-9% of the initial binding capacity. In contrast, in pair-fed controls, the mucin binding to MBP remained the same or increased up to 20%. Results of the studies, performed on mucin of the individual animals and matching preparations of MBP, showed that each animal expresses different degree of mucin binding. Moreover, in chronic ethanol ingestion, the individual variations are accompanied by a decrease in mucin binding to MBP. Since the observed decrease in binding occurred in samples containing the same preparation of MBP, the component affected by alcohol resides on mucin. Thus, considering the in vitro impact of ethanol on generation of carbohydrate chains in Golgi, and the finding on mucin oligosaccharides-dependent mucin-MBP complex formation, we conclude that ethanol impairs the synthesis of mucin oligosaccharide structures required for binding with MBP, and the retention on gastric mucosal surfaces.     

Platelet-derived growth factor activation of gastric mucosal calcium channels.

Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhancement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis.     

Role of mucosal injury in initiating recurrent aphthous stomatitis.

The buccal mucosa of 30 patients with recurrent aphthous stomatitis and 15 healthy controls was injured by suture and penetration with a tenaculum and a hypodermic needle and each of the six puncture wounds produced monitored for up to seven days for the development of ulcers. Altogether 26 lesions were induced in 13 patients, whereas none occurred in the controls (p less than 0.001). Sutures caused most of the lesions (15), and those so induced had a mean maximum diameter of 2.3 mm and lasted for an average of four days. Ulcers induced mechanically were clinically indistinguishable from those usually seen in the patients, except that they were generally smaller and healed more quickly. These findings confirm that mechanically induced injury of the oral mucosa may cause ulceration in people susceptible to aphthous stomatitis. Such a procedure may therefore be helpful in identifying subsets of patients.     

Ischemic injury to rat forebrain mitochondria and cellular calcium homeostasis.

The three-vessel occlusion model of Kameyama et al. (Kameyama, M., Suzuki, J., Shirane, R. and Ogawa, A. (1985) Stroke 16, 489-493) was adapted with modifications to induce complete reversible rat forebrain ischemia. A fast and simple procedure for the isolation and purification of rat brain mitochondria, which provides high yield, is described. Mitochondria isolated from ischemic brain (12-30 min ischemia) exhibited decreases in State 3 respiratory rates of approx. 70% with NAD-linked respiratory substrates. Less effect was observed with succinate and rotenone. The State 4 respiratory activity remained near control levels except at 15 min of ischemia (25% increase) with NAD-linked substrates. Similarly, with succinate and rotenone, an approx. 30% increase in State 4 activity was observed at 20 min of ischemia. Consequently, the respiratory control indices (RCIs) were decreased. Both the respiratory rates and RCIs could be restored to near control levels upon the addition of EGTA(EDTA) or ruthenium red to the assay mixture. Analysis employing fura-2 as a Ca2+ probe, indicated a great decrease in the first order rate constant for Ca2+ uptake of ischemic mitochondria and a significant increase in Ca2+ homeostasis with an increase in the cytosolic Ca2+ concentration which results in excessive association of Ca2+ on the mitochondrial membrane and an inhibition of the respiratory chain-linked oxidative phosphorylation and Ca(2+)-transport activity of forebrain mitochondria. These deficits are proportional to the duration of ischemia.     

Some new aspects of gastric mucosal protection and damage

Much of the research on gastric mucosal protection has concerned prostaglandins. Some of the recent studies consolidate aspects first investigated a few years ago, but whose importance is now becoming established more clearly. This short review will mention some of the more recent work demonstrating the importance of prostaglandins in preventing stasis of gastric mucosal blood flow, effects on cell senescence and exfoliation, and the protection of a severe mucosal lesion by a mucus-containing plug which facilitates healing. The leukotrienes are other substances formed in the gastric mucosa from the same precursors as the prostaglandins. Their roles are not well understood, but may include participation in gastric inflammation, and in mucosal damage by nonsteroidal anti-inflammatory drugs (NSAIDs) and ethanol. The NSAIDs may damage the gastric mucosa not only by reducing the formation of protective prostaglandins, but also by increasing the metabolism of prostaglandin precursors into leukotrienes. Another factor is thromboxane A2, a substance that is damaging to the gastric mucosa but whose synthesis is inhibited by NSAIDs. The prostaglandin analogues produced for the treatment of peptic ulcer may find a major use in the protection against damage by NSAIDs. Not only may they act as 'replacement therapy' for the inhibited prostaglandins, but they protect against damage from substances that do not inhibit prostaglandin synthesis. In doses that raise the gastric pH, the prostaglandins reduce the local absorption of NSAIDs by increasing their ionisation. In rats, paracetamol protects against damage by aspirin, but whether this occurs in man is controversial. Work not previously published demonstrates that paracetamol does not affect the inhibition of prostaglandin formation by indomethacin in human isolated gastric mucosa.     

Role of mucus in mucosal protection through ethanol and pepsin damage models.

Gastrointestinal mucus is considered an important part of the mucosal defence mechanism against endogenous aggressors such as acid and pepsin. The mucus gel layer, adherent to the mucosal surface creates a diffusion barrier to luminal pepsin, thus protecting the underlying epithelium from the digestion by pepsin. The mucolytic pepsin will, however, digest the mucus at its luminal surface, but that lost is normally balanced by secretion of new mucus. This dynamic balance is disrupted when the mucus is exposed to excess pepsin, which causes focal haemorrhagic damage by progressively hydrolyzing the adherent mucus. The adherent mucus gel layer cannot contribute to the protection against exogen damaging agents such as ethanol and nonsteroidal anti-inflammatory drugs, as these compounds easily penetrate the mucus barrier causing, at high concentration, epithelial exfoliation. This study describes the basic properties and characteristics of gastric mucus and compares the pepsin-induced damage with the ethanol damage model.     

Role of high-voltage-activated calcium channels in glucose-regulated beta-cell calcium homeostasis and insulin release

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, L(C)-type, and L(D)-type) in rat pancreatic beta-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Role of the polymeric Ig receptor in mucosal B cell homeostasis

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR(-/-) mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR(-/-) mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR(-/-) mice had approximately 3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA(+) B220(-) phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR(-/-) mice. Subsequent experiments showed that OVA-specific CD4(+) T cell expansion following OVA feeding was not elevated in pIgR(-/-) mice. Furthermore, no differences in CD8(+) T cell tolerance or induction of influenza virus-specific CD8(+) T cells were detected in pIgR(-/-) mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.     

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca²⁺]_i. However, the mechanism of silica- induced cytotoxicity relative to [Ca²⁺]_i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca²⁺]_i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca²⁺]_i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca²⁺]_o markedly inhibited the [Ca²⁺]_i rise (≈90%), suggesting that Ca²⁺ influx from extracellular source is a major mechanism for silica-induced [Ca²⁺]_i rise. When used at low doses but sufficient to cause a transient [Ca²⁺]_i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca²⁺]_o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca²⁺ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca²⁺ influx is inhibited by the Ca²⁺ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca²⁺ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca²⁺]_i rise was not due to the impairment of Ca²⁺-pumps, which restricts Ca²⁺ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca²⁺]_i rise. Taken together, these results suggest that the elevation of [Ca²⁺]_i caused by silica is due mainly to Ca²⁺ influx through plasma membrane Ca²⁺ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca²⁺ leakage during phagocytosis was attributed to the silica-induced [Ca²⁺]_i rise. © 1993 Wiley-Liss, Inc.