Protein kinase C enhances Ca2+ mobilization in human platelets by activating Na+/H+ exchange

Control of cytoplasmic pH (pHi) by a Na+/H+ antiport appears a general property of most eukaryotic cells. In human platelets activation of the Na+/H+ exchanger enhances Ca2+ mobilization and aggregation induced by low concentrations of thrombin (Siffert, W., and Akkerman, J. W. N. (1987) Nature 325, 456-458). Several observations indicate that the exchanger is regulated by protein kinase C. (i) Inhibitors of protein kinase C (trifluoperazine, sphingosine) inhibit the increase in pHi seen during thrombin stimulation as well as Ca2+ mobilization; artificially increasing pHi by monensin or NH4Cl then restores Ca2+ mobilization. (ii) Direct activation of protein kinase C by 1-oleoyl-2-acetylglycerol initiates an increase in pHi that depends on the presence of extracellular Na+ and is sensitive to inhibition by ethylisopropylamiloride. The pHi sensitivity of thrombin-induced Ca2+ mobilization is particularly evident in the range between pH 6.8 and 7.4 and at low thrombin concentrations, whereas thrombin concentrations of more than 0.2 unit/ml bypass the pH sensitivity. In the absence of thrombin an increase in pHi, either induced artificially (by addition of the ionophores nigericin or monensin) or via activation of protein kinase C (by addition of 1-oleoyl-2-acetylglycerol), does not induce Ca2+ mobilization. We conclude that activation of protein kinase C is essential for Ca2+ mobilization in platelets stimulated by low concentrations of thrombin and that protein kinase C exerts this effect via activation of the Na+/H+ exchanger.     

Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets.

The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.     

Molecular link between membrane cholesterol and Na+/H+ exchange within human platelets.

Incubation of human platelets with cholesterol-poor, cholesterol-normal and cholesterol-rich liposomes revealed that: (i) acquisition or depletion of platelet membrane cholesterol was highly selective; (ii) variation in membrane cholesterol was highly selective. Variation in membrane cholesterol content (cholesterol-to-phospholipid molar ratio from 0.15-1.2) with respect to values found in unmodified normal platelets, was paralleled by the observed changes in amiloride-sensitive cytoplasmic pH, as well as phospholipase A2 activity. However, a decrease in cytoplasmic pH was accompanied by an increase in phospholipase A2 activity; (iii) membrane cholesterol-modulated changes in intra-platelet pH, as well as phospholipase A2 activity, was completely inhibited when platelets were pretreated with quinacrine (a specific phospholipase A2 inhibitor) before exposure to various types of liposomes. Although exposure of platelets (pretreated with amiloride) with various types of liposomes resulted in the inhibition of Na+/H+ exchange it had no noticeable effect upon the observed phospholipase A2 activity. Based upon these results we suggest that membrane cholesterol-modulated phospholipase A2 activity may be the basic mechanism responsible for the nature of Na+/H+ exchanger activity observed in cholesterol-enriched platelets, leading these platelets to a hypersensitized state.     

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.     

Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Modulation of Na+/H+ exchange activity by Cl-

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.     

Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.     

Na+/H+ exchange is not necessary for protein kinase C-mediated effects in platelets

The role of Na+/H+ exchange in protein kinase C-mediated effects in platelets was investigated by studying the effect of removal of extracellular Na+ ([Na+]e) on the different responses induced by phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoylglycerol (diC8). None of the responses studied, namely, protein phosphorylation, translocation of enzyme activity to the membrane fraction, potentiatory and inhibitory effect on platelet activation ([Ca2+]i, arachidonate and granule release) showed an absolute dependence on [Na+]e. With the exception of dense-granule release, which was clearly potentiated by the removal of [Na+]e and showed a negative correlation with exchanger activity, the other effects of PMA and diC8 were not affected by [Na+]e removal. It is concluded that Na+/H+ exchange is not essential for protein kinase C activation in platelets.     

Calcium-calmodulin modulation of the activity of Na+/H+ exchanger in human platelets

This study aimed at establishing the role of calmodulin in regulating pHi of human platelets under acid loads and in stimulated states. The response of human platelets to thrombin was an initial drop of pHi followed by a recovery with a significant increase above the pre-stimulation level in control experiments and a recovery to initial values in platelets maintained in the presence of 19 mmol/l TFP (trifluoperazine = 2 trifluoromethyl-10 [3'-(1 methyl-4-piperazinyl) propyl] phenothiazine). The change in pHi after 8 min was 0.130 +/- 0.030 in the control and 0.001 +/- 0.011 pH units in TFP (P < 0.05). The initial velocity of recovery from an acid load was reduced to 56.7 +/- 6% of the control (n = 6, P < 0.05) with 50 mumol/l W7 (N-(6 aminohexyl)-5-chloro-l-naphthalene sulphonamide), and to 29.7 +/- 4.3% of the control (n = 8, P < 0.05) with 19 mumol/l TFP. The initial velocity of recovery was significantly greater in recalcified platelets than in the preparations kept in the nominal absence of extracellular calcium (1.08 +/- 0.12 vs 0.66 +/- 0.12 pH units per min, P < 0.05). Lower concentration of TFP had an inhibitory effect only in the presence of calcium. The velocities of recovery reached similar values at higher TFP concentration. The significant interaction between Ca2+ and TFP concentrations indicates that the Ca-calmodulin complex, rather than an unspecified direct action of TFP, is responsible for the modulation of the Na+/H+ exchanger. These findings indicate that calcium-calmodulin participates in both the recovery of pH after an acid load and the increase of pHi in stimulated states of human platelets.     

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.     

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.     

Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids.

In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.     

Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+o/H+i exchange process from the matrix side of the membrane. The Na+-induced changes in the pH of the matrix (pHm) showed that: (i) the Na+o/H+i exchange followed Michaelis-Menten kinetics with respect to external Na+ with a Km of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+-induced alkalinization occurred above a pHm of 7.45 +/- 0.09 (n = 4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.     

Thrombin-induced platelet aggregation is affected by external Na+ independently of the Na+/H+ exchange

Thrombin affects blood platelets by activation of Na+/H+ exchange and induction of aggregation, but the relationship between these effects is under debate. The present study attempts to clarify whether the activation of the exchanger activity is required for platelet aggregation. In apparent support of such a requirement, thrombin-induced aggregation is higher in Na+ medium than in N-methylglucamine+ medium and is inhibited by sphingosine, an inhibitor of protein kinase C known to regulate the Na+/H+ exchanger. However, the inhibition of aggregation by sphingosine occurs in both Na+-containing and Na+-free media, the aggregation is identical in Na+ and K+-containing media, and is not inhibited by 5-N-(3-aminophenyl)amiloride, at a concentration 10-fold higher than its Ki for platelet Na+/H+ exchange. Furthermore, at low concentration (0.005 U/ml) thrombin induces aggregation but does not activate the exchange. It is concluded that the activation of Na+/H+ exchange is not required for thrombin-induced platelet aggregation and that the apparent augmentation of aggregation by Na+ is due to an inhibitory effect of N-methylglucamine+.     

Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors

The cytoplasmic pH (pHi) of human blood neutrophils was measured using trapped carboxyfluorescein derivatives. Cells were acid-loaded using propionate or by pretreatment with NH4+. Acid-loaded cells were found to regain near-normal pHi by means of a Na+-dependent process. A concomitant Na+ uptake was recorded as a change in cell volume. Both events were amiloride-sensitive, indicating involvement of a Na+/H+ antiport. Activation of Na+/H+ exchange was also observed with chemotactic factors. Studies of the pHi-dependence of the H+ extrusion rate indicate that chemotactic factors increase the [H+i] sensitivity of the antiport.     

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.