Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.     

Amiloride analogs stimulate protein phosphorylation and serotonin release in human platelets

We examined the effects of newly exploited amiloride analogs on protein phosphorylation and serotonin secretion in human platelets. 5-(N-methyl-N-isobutyl) amiloride (IBA) and, to a lesser extent, 5-(N-methyl-N-isopropyl) amiloride (IPA), highly specific inhibitors of Na+/H+-pump, induced the phosphorylation of 47K-dalton protein and myosin light chain (20K). The phosphorylation was inhibited by apyrase. On the other hand, 3', 4'-dichlorobenzamil (DCB) and 2', 4'-dimethylbenzamil (DMB), highly specific inhibitors of Na+/Ca2+-pump, and to a lesser extent amiloride analogs induced serotonin secretion. Apparently there was dissociation between the phosphorylation and the serotonin release induced by the analogs.     

The role of protein kinase C in T lymphocyte proliferation. Existence of protein kinase C-dependent and -independent pathways

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-2-induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+ anti-porter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two different pathways for T cell proliferation, one which involves protein kinase C and the other which does not.     

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.     

Amiloride, a specific inhibitor for the Na+-driven flagellar motors of alkalophilic Bacillus

Na+-driven flagellar motors of alkalophilic Bacillus were found to be inhibited by amiloride, a potent inhibitor for many Na+-coupled systems. A concentration of 0.5 mM of amiloride completely inhibited motility but showed almost no effect on the membrane potential, the intracellular pH homeostasis, and the ATP content of the cells. Furthermore, the activity of a Na+-coupled amino acid transport system was reduced only by half by this concentration of amiloride. Thus, the inhibition of motility of alkalophilic Bacillus by amiloride was rather specific. The inhibition of motility produced by amiloride was restored by increasing Na+ concentrations in the medium. Kinetic analysis of the data revealed that the inhibition was competitive with respect to the concentration of Na+ in the medium. Therefore, it is quite logical to assume that amiloride inhibits the rotation of the Na+-driven flagellar motors of alkalophilic Bacillus by competing with Na+ at the force-generating site of the motor. Some amiloride analogs known to selectively inhibit Na+ channels were potent inhibitors for the flagellar motors, suggesting that the Na+-interacting site of the motors has some similarity to that of the Na+ channels.     

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 1. Mechanism of inhibition by amiloride analogues

The mechanism by which terminal guanidino nitrogen substituted analogues of amiloride inhibit Na-Ca exchange in purified cardiac sarcolemmal membrane vesicles has been investigated. These inhibitors block both Nai-dependent Ca2+ uptake and Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange monitored in K+ is noncompetitive vs Ca2+ but competitive vs Na+. Substitution of sucrose for K+ results in mixed kinetics of inhibition vs Ca2+, suggesting a complex interaction between inhibitor and carrier under this condition. Amiloride derivatives also block two other modes of carrier action: Na-Na exchange is inhibited in a competitive fashion with Na+ and kinetics of Ca-Ca exchange inhibition are mixed vs Ca2+ in either sucrose or K+. However, Ca-Ca exchange inhibition can be alleviated by increasing K+ concentration. Dixon analyses of Na-Ca exchange block with mixtures of inhibitors suggest that these agents are interacting at more than one site. In addition, Hill plots of inhibition are biphasic with Hill coefficients of 1 and 2 at low and high inhibitor concentrations, respectively. These results indicate that amiloride derivatives are mechanism-based inhibitors that interact at two classes of substrate-binding sites on the carrier; at low concentration they bind preferentially to a site that is exclusive for Na+, while at higher concentration they also interact at a site that is common for Na+, Ca2+, and K+.     

Intracellular pH and cell adhesion to solid substrate

It was shown that activation of the Na+/H+ antiporter resulting in an increase of intracellular pH (pHi) by 0.2-0.3 is a necessary stage of cell stimulation by soluble growth factors. Solid substrate can also be formally regarded as a growth factor since adhesion stimulates proliferation of various cell types. In the present study we have found that the attachment of mouse embryo fibroblasts to solid substrate is followed by an increase of pHi by approx. 0.3 units. pH shift occurs after the cell attaches to the substrate and is obligatory for cell spreading. The evidence for Na+/H+ antiporter involvement in the increase of pHi in substrate-attached cells is presented. It is suggested that signals for cell proliferation by chemical (soluble ligands) and physical (solid substrate) growth factors are transmitted similarly.     

Wound currents and wound healing in the newt,Notophthalmus viridescens

Summary Wounded amphibian skin heals initially by a migration of epithelial cells from the cut edge towards the center of the wound. The density of currents leaving wounds made in Notophthalmus viridescens skin was manipulated in order to determine whether electrical fields associated with these currents might have a significant role in promoting this cell migration during wound healing. Wounds were made with either a needle (200 m) or a biopsy punch (500 m). Currents leaving the wounds were measured with a vibrating probe, and the wounds fixed at various times after wounding. When the Na⁺-dependent currents were reduced by blocking Na⁺ channels with benzamil, wound healing, as revealed by scanning electron microscopy and by paraffin histology, was impaired. These results are consistent with the hypothesis that there is an electrical component to wound healing.     

DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.     

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.