Amiloride and amiloride analogs inhibit Na+/K+-transporting ATPase and Na+-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na+-H+ exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na+/K+-transporting ATPase (Na+/K+-ATPase) and Na+-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased steady-state Na+ content and inhibited ouabain-sensitive 86Rb+ uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC50) of 3.0.10(-3) M, 5.2.10(-4) M, and 1.2.10(-4) M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC50 values of 2.2.10(-3) M, 2.2.10(-3) M, and 1.7.10(-4) M, respectively). In separate experiments, amiloride (5.10(-3) M), DMA (10(-3) M), and EIA (2.5.10(-4) M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10(-3) M) demonstrated that this inhibition was largely due to a decrease in the Na+-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na+/K+-ATPase directly, reversibly, and with a relative rank order potency of EIA greater than DMA greater than amiloride. All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na+-coupled transport processes as well.     

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.     

Control of intracellular pH and growth by fibronectin in capillary endothelial cells

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.     

The cellular pool of Na+ channels in the amphibian cell line A6 is not altered by mineralocorticoids. Analysis using a new photoactive amiloride analog in combination with anti-amiloride antibodies

An amiloride-sensitive Na+ channel is found in the apical plasma membrane of high resistance, Na+ transporting epithelia. We have developed a method for the identification of this channel based on the use of a new high affinity photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), and anti-amiloride antibodies to identify photolabeled polypeptides. NMBA specifically labels the putative Na+ channel in bovine kidney microsomes. A 130-kDa polypeptide is detected on immunoblots with anti-amiloride antibodies. NMBA is a potent inhibitor of Na+ transport in the established amphibian kidney epithelial cell line A6, and specifically labels a 130-kDa polypeptide. We utilized both NMBA photolabeling and [3H]benzamil binding in order to examine the cellular pool of putative channels following hormonal regulation of Na+ transport. This pool is not significantly altered by the mineralocorticoid agonist aldosterone or antagonist spironolactone, despite a 3.8-fold difference in transepithelial Na+ transport.     

[3H]ethylpropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+ exchange system. Its use with kidney cell membranes.

The interaction of amiloride and several amiloride derivatives with the Na+/H+ exchange system in Madin-Darby canine kidney cells and in rabbit renal microvillus membrane vesicles was studied from 22Na+ uptake experiments. On both types of preparation, the order of potency of the different molecules tested is: ethylisopropylamiloride greater than ethylpropylamiloride (EPA) greater than amiloride greater than benzamil. 3H-labelled EPA was prepared and used to titrate amiloride binding sites in solubilized microvillus membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]EPA and unlabelled EPA indicate that EPA recognizes a single family of binding sites with a Kd value of 45 nM and a maximum binding capacity of 2 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]EPA competition experiments is identical to that found for the inhibition of 22Na+ uptake by the Na+/H+ exchange system, suggesting that [3H]EPA binding sites are associated with the Na+/H+ exchange system. [3H]EPA binding sites are pharmacologically distinct from those of [3H]benzamil and [3H]bumetanide in kidney membranes.     

Amiloride-sensitive Na+ pathways in capillary endothelial cell swelling during hemorrhagic shock.

We recently discovered that the endothelium of skeletal muscle capillaries swells in the low-flow ischemia induced by hemorrhagic shock. The present study was undertaken to determine the Na+ transmembrane pathways involved in this swelling, since hypoxic cell swelling is attributed to an influx of Na+ and water. In an initial series of experiments, amiloride (5 mg/kg body wt), which blocks multiple Na+ pathways, was infused intravenously into anesthetized rabbits 30 min prior to shock (40% single-withdrawal hemorrhage). Intravital microscopy of treated capillaries in the rabbit tenuissimus muscle showed that after a 1-h shock period, there was no endothelial cell swelling, as evidenced by no measurable change in the width of red blood cells traversing the capillary. In contrast, the swollen endothelium of untreated capillaries reduced the luminal diameter by 20-25% with a preserved stationary abluminal membrane. The specific effects of amiloride on Na+ transport were investigated with amiloride analogues. Animal pretreatment with 5-(N,N-hexamethylene)amiloride, a selective inhibitor of Na(+)-H+ activity, in a dose of 0.5 mg/kg did not significantly mitigate shock-induced swelling; however, a dose of 1 mg/kg completely prevented it. Phenamil, a selective inhibitor of Na+ channel conductance, even at a potent dosage of 0.5 mg/kg, did not affect swelling. These results suggest a primary role for Na(+)-H+ exchange in endothelial cell swelling during hemorrhagic shock, possibly as a means to regulate cellular pH, which may become acidic during ischemia. Narrowed capillaries with elevated hydraulic resistances could delay and diminish resumption of microcirculatory flow on shock resuscitation.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.