Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells

Suspension-cultured cells of Lycopersicon peruvianum L. reacted to the presence of mechanically damaged cells with a transient alkalinization of their culture medium. This response resembled the alkalinization observed after treatment with fungal signal molecules such as chitin fragments and ergosterol or after application of the protein phosphatase inhibitor calyculin A. When compounds implicated in wound signalling were tested, the 18 amino acid peptide systemin was found to be a potent inducer of the alkalinization response, with a half-maximal activity at concentrations of ～100 pM. The decrease in extracellular H⁺ was paralleled by an increase of K⁺, and induction of both ion fluxes was blocked by the protein kinase inhibitor K-252a. Systemin also caused rapid increases in the activities of 1-aminocyclopropane-1-carboxylate (ACC) synthase and phenylalanine ammonia-lyase, two other responses commonly observed in cells treated with elicitors. The systemin analogue systemin-Ala¹⁷, a reported systemin antagonist in the induction of proteinase inhibitors in tomato plants, provoked a much weaker alkalinization response and did not induce ACC synthase at all. When applied together with authentic systemin, this analogue antagonized induction of both responses, indicating that the perception system for systemin had very similar properties in the L. peruvianum cells as in tomato plants. In conclusion, suspension-cultured L. peruvianum cells provide a convenient and highly sensitive system to study elements of wound response and, in particular, systemin perception.     

Perception of Fungal Sterols in Plants (Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells)

Suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) reacted to spores and spore exudates of the pathogen Cladosporium fulvum with a rapid, transient alkalinization of their growth medium that resembled the previously described alkalinization response elicited by chitin fragments (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316) and was likewise inhibited by the protein kinase inhibitor K-252a. However, the spore factor recognized by the cells differed from chitin fragments in that it was butanol soluble and active in cells refractory to stimulation by chitin fragments. The spore factor was purified and identified as ergosterol, the main sterol of most higher fungi. With pure ergosterol, half-maximal induction was reached at about 10 pm. After treatment with ergosterol, tomato cells became refractory to a subsequent stimulation by C. fulvum and vice versa, indicating that ergosterol was the principal component of the spores recognized by the plant cells. Most other sterols were inactive, including cholesterol, a range of animal steroid hormones, and all natural plant sterols tested, except for stigmasterol, which was about 106 times less active than ergosterol. Our data demonstrate that tomato cells perceive ergosterol with a selectivity and sensitivity that resembles the perception of steroid hormones in animals.     

Desensitization of the Perception System for Chitin Fragments in Tomato Cells

Suspension-cultured tomato (Lycopersicon esculentum) cells react to stimulation by chitin fragments with a rapid, transient alkalinization of the growth medium, but behave refractory to a second treatment with the same stimulus (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307–316). We analyzed this phenomenon and found that chitin fragments caused desensitization in a time- and concentration-dependent manner. Partially desensitized cells exhibited a clear shift toward lower sensitivity of the perception system. The ability of chitin oligomers to induce desensitization depended on the degree of polymerization (DP), with DP5 ≈ DP4 DP3 DP2 > DP1. This correlates with the ability of these oligomers to induce the alkalinization response and to compete for the high-affinity binding site on tomato cells and microsomal membranes, indicating that the alkalinization response and the desensitization process are mediated by the same receptor. The dose required for half-maximal desensitization was about 20 times lower than the dose required for half-maximal alkalinization; desensitization could therefore be used as a highly sensitive bioassay for chitin fragments and chitin-related stimuli such as lipochitooligosaccharides (nodulation factors) from Rhizobium leguminosarum. Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of high-affinity binding sites from the cell surface, and thus appears to be caused by an intermediate step in signal transduction.     

Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study.

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.     

IL-1 activates the Na+/H+ antiport in a murine T cell

One of the early events following growth factor exposure is elevation of intracellular pH, a process mediated by the Na+/H+ antiport. We studied the effects of human rIL-1 alpha (HrIL-1 alpha) on intracellular pH (pHi) and calcium ([Ca2+]i) in a murine T cell line (MD10 cells), which proliferates in response to IL-1 alone. By using the intracellularly trapped fluorescent dyes (2(1),7(1)-bis-2-carboxyethyl)-5(and -6) carboxyfluorescein) and indo-1, we monitored immediate to early changes of pHi and [Ca2+]i in response to HrIL-1 alpha. Exposure to HrIL-1 alpha (120 pM) leads to an early, sustained intracellular alkalinization (delta pH = + 0.09 +/- 0.03) that plateaus within 20 min. Lower concentrations of the monokine (12 pM, 1.2 pM) have a positive but not statistically significant effect on pHi. These effects parallel the degree of MD10 IL-1R saturation predicted by the KD (49 pM) as assessed by 125I-HrIL-1 alpha binding by MD10 cells (Bmax = approximately 1300). Both the MD10 IL-1 receptor KD and the HrIL-1 alpha concentration required to induce early measurable alkaline pH shifts, however, exceed by three orders of magnitude the HrIL-1 alpha ED50 (50 fM) required for MD10 proliferation. The IL-1-induced rise in pHi is both sodium dependent and amiloride sensitive, indicative of activation of the Na+/H+ antiport. Additionally, PMA (100 nM) and IL-2 (2 nM) alkalinize MD10 cells, with the rise in pHi as a result of PMA exceeding the maximal IL-1 effect (delta pH = + 0.13 +/- 0.04). Furthermore, although PMA alkalinizes cells previously exposed to HrIL-1 alpha, the monokine does not alter the pHi of PMA-treated MD10 cells. Importantly, intracellular alkalinization induced by either HrIL-1 alpha or PMA is inhibited by staurosporine (1 mu iM). Finally, HrIL-1 alpha does not change MD10 [Ca2+]i, in either an acute or sustained fashion. These results indicate that IL-1 activates the Na+/H+ antiport in T cells by a mechanism that is unrelated to changes in [Ca2+]i but may involve protein kinase C activation.     

Alfalfa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L.) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst. Received: 23 November 1998 / Accepted: 23 June 1999     

Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism

In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.     

Evaluation of a chromogenic chito-oligosaccharide analogue, p-nitrophenyl-β-D-N,N'-diacetylchitobiose, for the measurement of the chitinolytic activity of bacteria

Three methods of quantifying chitinase activity were compared. The activities of crude chitinases of 10 bacterial isolates from different environments were estimated in terms of (1) the release of p -nitrophenol from the chromogenic chito-oligosaccharide analogues, p -nitrophenyl-β-D- N,N' -diacetylchitobiose, p -nitrophenyl- N -acetyl-β-D-glucosamine and p -nitrophenyl-β-D- N,N',N" -triacetylchitotriose, (2) the release of reducing sugars from chitin and (3) the formation of clearing zones on chitin agar. When crude chitinase from Bacillus pabuli was used the hydrolysis of p -nitrophenyl-β-D- N,N' -diacetylchitobiose correlated well with the release of reducing sugars from chitin and the formation of clearing zones on chitin agar. However, when the activity of crude chitinases from the different bacterial isolates were compared no agreement was found between the hydrolysis of p -nitrophenyl-β-D- N,N' -diacetylchitobiose and the release of reducing sugars from chitin or the formation of clearing zones on chitin agar. It was concluded that the assay with chromogenic p -nitrophenyl chito-oligosaccharide analogues is not well suited for studies that compare the chitinase activity of different bacteria.     

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.     

Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+, bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after approximately 10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin-induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2(+)-dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin-induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C-mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenesis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.     

N,N-Diacetylchitobiose production from chitin by Vibrio anguillarum strain E-383a

Vibrio anguillarum strain E-383a, isolated from sea water, accumulated a considerable amount of N,N '-diacetylchitobiose when it was cultivated in a medium containing colloidal chitin. The maximum conversion of chitin to chitobiose was found to be 40·3%. The rate of chitobiose accumulation was accelerated after the cessation of bacterial growth. Small amounts of N -acetylglucosamine and N,N',N -triacetylchitotriose were also accumulated but no other saccharides were detected. These results may suggest that strain E-383a produces an exo-type chitinase which successively hydrolyses the glycosidic linkages of chitin into biose units. The exclusive accumulation of chitobiose by the bacterial cells may provide a new, selective method for the production of this substance.     

Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts

Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF-AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, 1) a fast alkalinization, reaching a maximum pH in approximately 2-5 min; 2) a slow alkalinization, reaching a maximum pH in 10-20 min; 3) a very slow alkalinization, not reaching a plateau pH within the measurement time; 4) no apparent change in pH during the measurement time; 5) an early transient acidification, followed by either a fast or slow alkalinization; and 6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of approximately 0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.     

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).     

Effect of Bordetella heat-labile toxin on perfused lung preparations of guinea pigs

In vitro effects of the Bordetella HLT on the isolated perfused lung and some other tissue preparations from guinea pigs were examined. When HLT (30 to 300 MNDs/ml) was administered, an increase of the perfusion pressure was induced in the perfused lungs, indicating vasoconstriction. When 100 or 300 MNDs/ml of HLT was given, the pressure increase appeared after a lag period of 3.5 to 4 min, reached a maximum within 8 to 13 min, and then slowly decreased by 60 to 80% 25 min after exposure. In calcium-free medium, the pressure increase due to HLT did not occur, but these HLT-treated lungs manifested an increase without any lag period immediately after the calcium-free medium was replaced by normal medium containing calcium. No difference in the response of the perfused lungs to histamine was observed before and after exposure to HLT. The arterial strip did not respond to HLT, but after predigestion with a collagenase and elastase solution the contractive response to 100 MNDs/ml of HLT appeared with a lag period of 1 min. HLT had no effect on the pharmacological responses of the isolated atria, deferent canal or intestinal preparations, or on the ciliary movement of cultured tracheal rings.     

Rapid alkalinization in alfalfa root hairs in response to rhizobial lipochitooligosaccharide signals

Rhizobial lipochitooligosaccharides (Nod factors) function as symbiotic signals that trigger root hair deformations and cortical cell divisions on the roots of leguminous plants in a host-specific manner. By using pH-sensitive microelectrodes, it is shown that alfalfa root hair cells respond to Rhizobium meliloti Nod factors with a rapid intracellular alkalinization of 0.2–0.3 pH units. This alkalinization remained as long as the Nod factor was present, but slowly reversed after removal of the signal. The response was most sensitive to the sulfated tetrameric Nod factor, NodRm-IV(C16:2,S), which is morphogenic on the host plant alfalfa, suggesting a role in a signal transduction cascade. Non-sulfated Nod factor as well as chitooligosaccharides elicited a pH_c change only at elevated concentrations. The increase of PH_c in response to sulfated Nod factor was concomitant with a depolarization of the plasma membrane potential whereas the PH_c change in response to non-sulfated Nod factor occurred in the absence of membrane depolarization. In addition, whereas a first dose of sulfated Nod factor inhibited the subsequent response to a second dose of the same molecule, it did not significantly repress the activity of non-sulfated Nod factor. These results indicate that sulfated and non-sulfated Nod factors act independently and suggest the existence of two Nod signal perception systems, one transmitting the host-specific signal, the other representing an ancient reception system for a generic Nod factor structure.     

Dependence of the growth of pH-sensitive mutants of a facultatively alkalophilic Bacillus on the regulation of cytoplasmic pH

Abstract A facultatively alkalophilic Bacillus strain, YN-2000, is able to grow over a wide pH range, from 7.0–10.5, and was suggested to regulate its cytoplasmic pH by alkalinization and acidification systems at neutral and alkaline pH, respectively [9]. Two kinds of pH-sensitive mutants were isolated after treatment with N -methyl- N '-nitro- N -nitrosoguanidine. One mutant was neutral-sensitive and unable to grow below pH 8. The other was alkaline-sensitive and unable to grow above pH 9. The neutral- and alkaline-sensitive mutants were suggested to be unable to regulate cytoplasmic pH at neutral and alkaline pH, respectively.     

Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation

Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O₂ concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.     

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N'-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.     

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.